Prolactin synthesis was investigated in monolayer cultures of dispersed pituitary cells maintained in defined medium. Electrophoresis of cell extracts from cultures labeled with [35S]methionine demonstrated that treatment with the dopaminergic drug, ergocryptine, specifically inhibited prolactin synthesis. Analysis of the time course of ergocryptine effects demonstrated that prolactin synthesis decreased sharply after 1 to 2 days of treatment and appeared to reach a new level of 26 to 28% of control values after 4 to 6 days of treatment. The concentration of ergocryptine which produced half-maximal inhibition of prolactin synthesis was about 0.3 nM. The inhibition of prolactin synthesis produced by ergoctryptine treatment was reversed by removal of the ergocryptine. Dopamine, ergocryptine, and bromoergocryptine (also a dopamine agonist) all inhibited prolactin synthesis while epinephrine and norepinephrine had little effect. The concentration of prolactin mRNA in cultured cells was assayed by hybridization of total cell RNA to prolactin cDNA. Analysis of cultures treated either for varying times or with varying doses of ergocryptine demonstrated that there was a close correspondence between the inhibition of prolactin synthesis and the inhibition of prolactin mRNA levels. These studies demonstrate the ability of dopaminergic stimulation to specifically inhibit prolactin synthesis and to decrease prolactin mRNA levels in primary cultures of pituitary cells maintained in defined medium.