A widely distributed Ca2+- and phospholipid-dependent protein kinase, protein kinase C, may play a major role in cellular regulation. We now report that arachidonate can directly activate protein kinase C from human neutrophils. Activation was Ca2+-dependent and was enhanced by diolein, but did not require phosphatidylserine. Arachidonate enhanced the apparent affinity of the kinase for Ca2+ in the presence of phosphatidylserine. Other unsaturated, but not saturated, fatty acids also activated protein kinase C. These results suggest a novel means of leukocyte activation and cellular regulation: arachidonate, which is released by ligand-receptor interactions in neutrophils and many other cell types, could function as a second messenger via activation and modulation of protein kinase C.