This study examined the intracellular mechanisms for the regulation of tyrosine hydroxylase in the tuberoinfundibular dopaminergic neurons of cycling female rats. It also evaluated the hormonal influences that contribute to the control of this enzyme on proestrus. Tyrosine hydroxylase messenger RNA (mRNA) levels in the arcuate nucleus of the hypothalamus were assessed by in situ hybridization. Tyrosine hydroxylase activity in the stalk-median eminence was determined from the in vitro or in vivo rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation after inhibiting DOPA decarboxylase with brocresine or m-hydroxybenzylhydrazine, respectively. Tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation were similar on diestrous day 2 and proestrous mornings, but were reduced by 50% on estrus. Although circulating PRL concentrations were similar on the morning of each day of the estrous cycle, a broad preovulatory PRL surge was observed on the afternoon of proestrus. In vitro DOPA accumulation was similar at 1000 h before the PRL surge and at 1330 h during the peak phase of the PRL surge, but was reduced during the plateau phase of the PRL surge (1700 and 2200 h) coincident with the preovulatory progesterone rise and remained low on estrus. However, in vivo DOPA accumulation was transiently decreased only at 1700 h on proestrus. Tyrosine hydroxylase mRNA levels were similar at 1000, 1330, and 1700 h on proestrus, were reduced by 50% at 2200 h on proestrus subsequent to the decrease in enzyme activity, and remained low on the morning of estrus. Okadaic acid, a protein phosphatase-1 and -2A inhibitor, induced a similar increase in tyrosine hydroxylase activity in vitro at 1330 and 2200 h on proestrus and at 1100 h on estrus, indicating that tyrosine hydroxylase was capable of being activated in spite of decreased mRNA levels. Ovariectomy between 1100-1200 h on proestrus prevented the decrease in tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation at 2200 h. The effects of ovariectomy were completely reversed by progesterone, whereas estradiol had no effect. Circulating PRL levels at 2200 h were suppressed to basal levels after ovariectomy, but were increased by progesterone treatment at 1530 h to levels similar to those in the plateau phase of the PRL surge in control rats. Administration of the progesterone antagonist RU486 at 1200 h on proestrus did not alter tyrosine hydroxylase activity, tyrosine hydroxylase mRNA levels, or circulating PRL concentrations at 2200 h.(ABSTRACT TRUNCATED AT 400 WORDS)