Amphetamine is taken up through the dopamine transporter in nerve terminals and enhances the release of dopamine. We previously found that incubation of rat striatal synaptosomes increases phosphorylation of the presynaptic neural-specific protein, neuromodulin (Gnegy et al., Mol. Brain Res. 20:289-293, 1993). Using a state-specific antibody, we now demonstrate that incubation of rat striatal synaptosomes with amphetamine increases levels of neuromodulin phosphorylated at ser 41 , the protein kinase C substrate site. Phosphorylation was maximal at 5 min at 37Β°C at concentrations from 100 nM to 10 Β΅M amphetamine. The effect of amphetamine on the phosphorylation of synapsin I at a site specifically phosphorylated by Ca 21 /calmodulin-dependent protein kinase II (site 3), was examined using a state-specific antibody for site 3-phosphosynapsin I. Incubation with concentrations of amphetamine from 1 to 100 nM increased the level of site 3-phospho-synapsin I at times from 30 sec to 2 min. The effect of amphetamine on synapsin I phosphorylation was blocked by nomifensine. The presence of calcium in the incubating buffer was required for amphetamine to increase the level of site 3-phospho-synapsin I. The amphetamine-mediated increase in the content of phosphoser 41 -neuromodulin was less sensitive to extrasynaptosomal calcium. The amphetamine-mediated increase in the content of site 3-phospho-synapsin I persisted in the presence of 10 Β΅M okadaic acid and was not significantly altered by D1 or D2 dopamine receptor antagonists. Preincubation of striatal synaptosomes with 10 Β΅M of the protein kinase C inhibitor, Ro-31-8220, blocked the amphetamine-mediated increases in the levels of both phosphoser 41 -neuromodulin and site 3-phospho-synapsin I. Our results demonstrate that amphetamine can alter phosphorylation-related second messenger activities in the synaptosome.