Progesterone has the capacity to suppress hypothalamic dopaminergic neuronal activity on proestrous afternoon and prolong or amplify the preovulatory prolactin surge in rats. In the present study, we examined enzyme activity and phosphorylation state of tyrosine hydroxylase (TH) in the stalk-median eminence (SME) of cycling female rats on proestrus and estrus, and related these to circulating progesterone levels. Phospho-TH levels were evaluated by western blot analysis. TH activity was determined from the rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation. Phospho-TH levels at Ser-19, Ser-31 and Ser-40 were similar at 1100, 1300 and 1500 h on proestrus, but declined at 1700, 1900 and 2200 h, coincident with rising serum progesterone levels. Similarly, DOPA accumulation was 30–50% lower at 1700, 1900 and 2200 h as compared to 1100–1500 h on proestrus. Ser-31 and Ser-40 phosphorylation state was increased by 1100 h on estrus to a level similar to 1100 h on proestrus, whereas DOPA accumulation was 30% greater on estrous as compared to proestrous morning. There were no significant differences among the several time points on proestrus and estrus with regard to TH protein or -tubulin levels. Exogenous progesterone administration (7.5 mg/kg, s.c.) prior to the preovulatory progesterone surge decreased TH activity and phospho-TH at Ser-19, Ser-31 and Ser-40, accompanied by premature increased serum prolactin. Our study suggests that decreased TH phosphorylation at Ser-19, Ser-31 and Ser-40 contributes to the decline in TH activity in the SME on proestrous afternoon and that progesterone may cause this initial cytoplasmic response of TH dephosphorylation.