A number of cellular responses mediated by protein kinase C (PKC) in the anterior pituitary gland involve a form of PKC that is unusually resistant to the PKC inhibitor H7. In this study, inhibition by staurosporine, H7 and Ro 31-8220 of phorbol 12,13-dibutyrate-induced PKC activity in cytosol partially-purified from rat midbrain, anterior pituitary and a number of other tissues, as well as COS 7 cells, was studied. An in vitro mixed micelle histone IIIS phosphorylation assay was used to allow comparison of Ca²⁺-dependent and Ca²⁺-independent PKC activity. In anterior pituitary but not midbrain or COS 7 cells, Ca²⁺-independent activity was notably resistant to H7 but sensitive to staurosporine and Ro 31-8220. All Ca²⁺-dependent activity was sensitive to these three inhibitors. Comparison of PKC activity from a variety of different tissue sources showed H7-resistant, Ca²⁺-independent activity also occurred in lung but not in spleen, thalamus or cerebellum. Mezerein and 1,2-dioctanoyl-sn-glycerol also activated this H7-insensitive PKC from anterior pituitary. Phosphorylation of a number of substrates by anterior pituitary PKCs were also compared. When [Ala^9,10,Lys^11,12] glycogen synthase_1-12 (GS peptide) but not [Ser]²⁵ PKC a_19-31 was the phosphate acceptor, a clear component of the activity, which was Ca²⁺-independent, was resistant to H7. The tissue distribution of this activity and its characteristic resistance to H7 but not other inhibitors, does not obviously correlate with that of any of the well-characterised PKCs, and may reflect either a novel or a modified isoform. Analysis of clones using selected restriction endonucleases and subsequent DNA sequencing analysis identified sequences encoding PKC α, and ζ but not novel PKC related sequences.