We studied the in vitro effects of omega-3 fish oils and other fatty acids on the activity of crude protein kinase C from S49 lymphoma cells, on partially purified enzyme from rat cerebrum, on homogeneous protein kinase C from bovine brain, and, for comparison, on type I adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In the absence of exogenous phospholipid, the fish oils cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and acid (DCHA) enhance the catalytic cis-4,7,10,13,16,19-docosahexaenoic activity of protein kinase C and support the binding of [3H]phorbol 12,13-dibutyrate, both to approximately 50% of the level supported by phosphatidylserine. In the presence of phosphatidylserine, the omega-3 fatty acids reduce catalytic activity and [3H]phorbol 12,13-dibutyrate binding by about one-half. The effects of the omega-3 fatty acids on enzyme activity suggest that fish oils act as partial agonists competitively with phosphatidylserine. EPA, DCHA, and arachidonate (but not a variety of saturated fatty acids) inhibit the cAMP-dependent protein kinase. Thus dietary fish oils and cellular fatty acids mobilized by the action of phospholipase A2 may differentially modulate the activities of protein kinase C and cAMP-dependent protein kinase. These data suggest means by which unsaturated fatty acids mobilized within cells may act as second messengers.