The regulatory pathways involved in the rapid response of the AP-I transcription factor component, c-fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MO-63 bone cell line were investigated using a four-point bending model. HOB and MO-63 cells showed up regulation of c-fos expression after I-hour loading on fibronectin and collagen type I substrates; however, MO-63 cells did not respond on laminin YIGSR substrates. It was suggested that RGD mediated integrin interactions might be non-essential for the mechanical induction of c-fos at certain time points as indicated by a lack of inhibition associated with ROD-peptide treatment (however, evidence of the inhibitory nature of soluble RGD-peptides at these time points may require further examination). β1 integrin mediated interactions are critical as induction was completely blocked by anti-β1 integrin antibodies. The role of calcium signalling pathways was demonstrated by blocking up regulation with addition of EGTA, which chelates extracellular Ca2+, and gadolinium, which inhibits stretch-activated channels. L-type calcium channels may also be contributory but not essential, as addition of the L-type channel blocker, nifedipine inhibited the response, but not completely. Further evidence for involvement of calcium pathways followed addition of the Ca2+ ionophore A-23187 that induced temporally identical up regulation without loading. Protein kinase (C) mediated pathways were also shown to be critical, as shown by abolition of the response following H7 -dichloride treatment. Prostaglandin signalling pathways were non-essential, but were implicated in maximising load induction of c-fos as indicated by indomethacin induced inhibition. C-fos promoter analysis indicated that the major CRE is not essential for mechanically induced transcriptional activation of c-fos. An SRE containing promoter fragment appears to play a key role in this induction but is also not essential, indicating that multiple response elements are required. These results therefore demonstrate the essential nature of calcium, integrin and protein kinase mediated pathways in the induction of the early transcriptional mechanoresponse of bone, which are mediated by multiple response elements in the c-fos promoter.