L-3,4-Dihydroxyphenylalanine (DOPA) is a precursor of a neurotransmitter dopamine (DA), and synthetized by tyrosine hydroxylase in the cytoplasm of catecholaminergic neurons. We proposed that DOPA is a neurotransmitter. Neurotransmitter storage and release are regulated by vesicular transport system. However, it is unknown whether there are vesicular transport system(s) for DOPA. In this study, we examined whether depolarizing stimuli release DOPA from cultured PC12 cells. Then we performed characterization of the evoked release of DOPA and DA. High K⁺(45 mM) induced the release of DOPA and DA. Both the releases were decreased by deprivation of extracellular Ca²⁺. We next examined the effects of bafilomycin, a vacuolar type H⁺-ATPase inhibitor, on the release, and found that bafilomycin inhibited the release of DA, but not DOPA release. In contrast, brefeldin A, which is known to induce Golgi complex di-assemble and its redistribution into the endoplasmic reticulum, thereby exerting its inhibitory action on secretion, suppressed the DOPA release and to a lesser extent DA release. These findings suggest that the release of DOPA may occur through a secretion pathway distinct from that for DA in PC12 cells.