Electrophoretic analysis of soluble proteins from pituitary cells pulse labeled with [35S]methionine demonstrated that 10 nM T3 inhibited PRL synthesis, but did not affect the synthesis of most other pituitary proteins. The effects of T3 were somewhat slow, requiring about 3 days for a 50% reduction in PRL synthesis. PRL synthesis slowly returned toward control levels after the removal of T3 from the culture medium. In serum-free medium, a concentration of about 0.6 nM T3 was required for half-maximal inhibition of PRL synthesis. In medium containing 5% fetal calf serum, only slightly higher concentrations of T3 were required to inhibit PRL synthesis. The Kd for the binding of [125]T3 to pituitary cell nuclei was 0.2 nM. Analysis of PRL mRNA levels by hybridization of total cellular RNA to PRL cDNA demonstrated that there was a good correspondence between T3 effects on PRL synthesis and PRL mRNA. These findings demonstrate that T3 can specifically inhibit PRL synthesis and PRL mRNA levels in cultured pituitary cells and suggest that T3 may have a physiological role in the regulation of PRL synthesis.