A variety of evidence indicates that activation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in nerve terminals leads to enhanced neurotransmitter release. Arachidonic acid and its 12-lipoxygenase metabolite, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), have been suggested to act as second messengers mediating presynaptic inhibition of neurotransmitter release. In the present study it was found that CaM-kinase II, purified from rat brain cortex, was inhibited both by arachidonic acid (IC50 = 24 microM) and by 12-HPETE (IC50 = 0.7 microM). Neither substance inhibited CaM-kinase I or III, protein kinase C, or the catalytic subunit of cAMP-dependent protein kinase. Specific inhibition of Ca2+/calmodulin-dependent protein phosphorylation by arachidonic acid was also demonstrated in intact synaptic terminals (synaptosomes) isolated from rat forebrain. These results suggest that arachidonate and its metabolites may modulate synaptic function through the inhibition of CaM-kinase II-dependent protein phosphorylation.