Free and membrane-associated fractions of protein kinase C (PKC) from rainbow trout (Onchorynchus mykiss) brain tissue were separated by hydroxylapatite chromatography and characterized kinetically. In both resting fish and in fish swum to exhaustion, approximately 40% of the total PKC activity was bound to membranes. Quantification of the three distinct hydroxylapatite chromatography peaks (PKC types gamma, beta and alpha) in cytosolic and membrane fractions revealed different isozyme distributions. The cytosolic fraction contained 21% PKC type gamma, 52% PKC type beta and 27% PKC type alpha. The membrane-associated fraction contained 23% PKC type gamma, 28% PKC type beta and 49% PKC type alpha. Kinetic characterization of the three isozymes showed that PKC type gamma was almost completely activated by Ca2+ alone whereas PKC type beta and PKC type alpha were 40% and 60% activated by Ca2+. Full activity for all enzymes was observed only in the presence of phosphatidylserine and diacylglycerol. Differences in the kinetic constants for the three isozymes were also apparent. PKC type gamma had a much lower affinity for Histone III-S when compared with PKC types beta and alpha (100 micrograms/ml as compared with 1.7 and 5.7 micrograms/ml). PKC type gamma also had a lower affinity for calcium (0.22 microM) when compared with PKC type beta (0.08 microM) and PKC type alpha (0.05 microM). PKC type alpha had a lower affinity for phosphatidylserine (8.6 micrograms/ml) when compared with PKC type gamma (0.37 microgram/ml) and PKC type beta (0.89 microgram/ml).