Src kinase is widely expressed in the brain and its inhibition with PP2 has previously been shown to depress depolarization-evoked glutamate release from rat cerebrocortical synaptosomes by reducing voltage-dependent Ca2+ entry. In this study, we further showed that the inhibitory effect of PP2 on 4-aminopyridine-evoked glutamate release results from a reduction of vesicular exocytosis and not from an inhibition of non-vesicular release. In addition, PP2 significantly inhibited ionomycin-induced or hypertonic sucrose-induced glutamate release. Also, disruption of cytoskeleton organization with cytochalasin D occluded the inhibitory action of PP2 on 4-aminopyridine and ionomycin-evoked glutamate release. These results suggest that PP2-mediated inhibition of glutamate release involves the modulation of some exocytotic steps, possibly through a regulation of actin cytoskeleton dynamics.