The role of tissue transglutaminase (tTGase), a calcium-dependent and GTP-modulated enzyme, in cell death processes is highly dependent on the type of stimuli. In this study, the role of tTGase in cell death was investigated using stably transfected fibroblast cell lines, which express tTGase in its active and inactive (Cys277Ser mutant) state. Two different cell death/stress stimuli were used: staurosporine (STS; a protein kinase inhibitor) and 3-nitropropionic acid (3-NP; an irreversible inhibitor of succinate dehydrogenase). In response to apoptotic stress induced by staurosporine, the transfected Swiss 3T3 clones inducible for the expression of catalytically active (TG3) and inactive (TGI 19) tTGase undergo apoptosis which appears to be ATP dependent. TG3 induced cells (- tet.) which express high levels of active tTGase had higher caspase3 activity than cells containing mutant inactive tTGase (TGI19). The site directed tTGase inhibitors Rob 281 and 283 not only inhibited tTGase, but also led to inhibition of caspase-3. Measured reduced tTGase activity during STS treatment confirmed previous findings that tTGase is a potential substrate of caspase and is cleaved during cells undergoing apoptosis. Inhibition of caspase activity by using the general caspase inhibitor (Z-VAD-FMK), did not prevent cell death, but did prevent degradation of tTGase. Tissue transglutaminase and cysteine proteases, which are both activated during apoptosis have a similar active site and several proteins act as common substrates for both cysteine proteases and tTGase during apoptosis. Changes in mitochondrial membrane potential are greater in transfected TG3 induced cells (- tet.) than their non-induced (+ tet.) controls. In the absence of tTGase protein, cells undergo apoptosis as measured by caspase-3 activation using tTGase null mouse embryonic fibroblasts (MEFs) (tTGase7'). No relevant differences were observed in wild type tTGase+/+ and tTGase7' cells. This suggests that following treatment of Swiss 3T3 fibroblasts with STS, tTGase is not involved in the central, essential apoptotic machinery, but may be involved in mitochondria function/dysfunction which is caspase independent. Cell stress induced by 3-nitropropionic acid (3-NP), resulted in a time dependent drop in ATP levels both in TG3 non-induced (+ tet.) and induced (- tet.) cells. However, measurement of the release of lactate dehydrogenase (LDH) into the culture medium indicated only a small change compared with untreated controls. In contrast, an increased presence of tTGase antigen could be detected in the extracellular matrix of cells induced to express the enzyme following 3-NP treatment, suggesting elevated tTGase secretion following cell stress. Measurement of in situ tTGase mediated cross-linking using the fluorescent primary amine substrate fluorescein cadaverine indicated a significant increase in amine incorporation in the extracellular matrix of the cells induced to express tTGase following 3-NP treatment. This cross-linking resulted in the formation of high molecular weight polymers in the extracellular matrix when deoxycholate insoluble proteins were analysed by SDS-PAGE. The data suggests that cell stress resulting from mitochondrial dysfunction results in the regulated release of tTGase which may be important in maintaining tissue integrity under these conditions. It also suggests that cells stressed in this manner, indicated by the substantial loss in ATP levels, release tTGase into the extracellular matrix where both its cross-linking and cell adhesion roles may be important in maintaining cell stability and tissue integrity. The results support the involvement of tTGase in a form of cell reaction to stress which does not fit the classical apoptotic or necrotic cell death process.