The site specificity of tyrosine hydroxylase phosphorylation in intact PC12 cells, labeled with 32Pi, was investigated. Digestion of 32P-tyrosine hydroxylase with trypsin produced five distinct 32P-labeled peptides (termed PC-1 through PC-5). Sequencing of the peptides revealed four acceptor sites: Ser8, Ser19, Ser31, and Ser40. The phosphorylation site in peptides PC-1 (AV-SEQDAK) and PC-2 (RAVSEQDAK) was identified as Ser19. Agents which cause calcium influx increased 32P incorporation into tyrosine hydroxylase at Ser19. PC-3 was identified as QAEAVTSPR, which contains the phosphorylation site Ser31. Nerve growth factor and phorbol dibutyrate increased 32P incorporation into Ser31. PC-4 was identified as the N-terminal amino acid sequence ((M)PTPSAPSPQPK), and the 32P incorporation occurred at Ser8. Of the agents tested, only okadaic acid (a protein phosphatase inhibitor) increased the phosphorylation of Ser8. PC-5 was shown to contain Ser40. Treatment of the PC12 cells with cAMP-acting agents increased 32P incorporation into Ser40. The present results demonstrate that some, but not all, of the phosphorylation sites demonstrated previously in vitro exist in situ. Conversely, the identification of Ser31 establishes a physiological phosphorylation site not previously reported in vitro. These four sites account for most, if not all, of the diversity in tryptic phosphopeptides reported previously for rat tyrosine hydroxylase.