Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-32P]ATP, in the presence or absence of 10 µM Ca2+, 1 µM cyclic AMP, 1 µM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+-stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+-stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.