We studied in vivo activity-dependent phosphorylation of tyrosine hydroxylase (TH) in dopaminergic (DA) neurons of the rat retina. TH phosphorylation (TH-P) was evaluated by immunocytochemistry, using antibodies specific for each of three regulated phosphorylation sites. TH synthesis rate was measured by dihydroxyphenylalanine (DOPA) accumulation in the presence of NSD-1015, an inhibitor of aromatic amino acid decarboxylase. TH-P was increased markedly by light or after intraocular injection of GABA(A) and glycine inhibitors. All three phosphospecific antibodies responded similarly to test drugs or light. A 30 min exposure to light increased DOPA accumulation by threefold over that seen after 30 min in darkness. Immunostaining to an anti-panNa channel antibody was found in all parts of the DA neuron. TTX blocked TH-P induced by light or GABA/glycine inhibitors but only in varicosities of the DA axon plexus, not in perikarya or dendrites. Veratridine increased TH-P in all parts of the DA neuron. The distribution of the monoamine vesicular transporter 2 was shown by immunocytochemistry to reside in varicosities of the DA plexus but not in dendrites, indicating that the varicosities are sites of dopamine release. Collectively, these data indicate that, in the retina, dopamine synthesis in varicosities is affected by the spiking activity of retinal neurons, possibly including that of the DA neurons themselves.