The snake venom phospholipase A2 neurotoxin, beta-bungarotoxin, acts presynaptically to alter acetylcholine release in both the peripheral and central nervous systems. In investigating the mechanism of this action, we found that beta-bungarotoxin inhibited phosphorylation of synapsin I, GAP-43 and MARCKS in rat brain synaptosomes. This inhibition was not due to the inhibition of ATP synthesis, action of arachidonic acid metabolites, or stimulation of phosphatase activities. Furthermore, the activities of Ca2+/calmodulin-kinase II, cAMP-kinase and protein kinase C were not altered by beta-bungarotoxin in either synaptic plasma membranes or cytosol. When synaptic plasma membranes were treated with beta-bungarotoxin, MARCKS phosphorylation was inhibited, and this inhibition was overcome by the addition of exogenous protein kinase C. These results suggest that the interaction between MARCKS and endogenous protein kinase C is altered by beta-bungarotoxin. In contrast, Naja naja atra phospholipase A2, a typical phospholipase A2 enzyme, had effects on phosphorylation which were different from those of beta-bungarotoxin: (1) inhibition of phosphorylation of synapsin I in intact synaptosomes was less potent than that by beta-bungarotoxin; (2) it stimulated basal phosphorylation of GAP-43 and MARCKS; and (3) it increased the activity of protein kinase C. The inhibition of synapsin I phosphorylation by N. n. atra phospholipase A2 in intact synaptosomes may be due to the inhibition of ATP synthesis. The stimulation of GAP-43 and MARCKS by N. n. atra phospholipase A2 can be explained by the production of arachidonic acid, which stimulated protein kinase C activity to a similar extent as that caused by N. n. atra phospholipase A2. Thus, the mechanism of action of beta-bungarotoxin appears to be quite different from that of a phospholipase A2 enzyme, suggesting that phospholipase A2 activity of beta-bungarotoxin may not be essential for its action. beta-Bungarotoxin may be a useful tool to study the physiological role of phosphorylation of synaptosomal proteins in neurotransmitter release.