The tripeptide thyroliberin (thyrotropin-releasing hormone, TRH) regulates the levels of cytoplasmic prolactin and growth hormone (somatotropin) mRNAs in a line of functional rat pituitary tumor cells. Intracellular mRNA levels were determined with specific complementary DNA probes in RNA-excess hybridization reactions and independently by two-dimensional gel electrophoretic analysis of the products of mRNA-directed cell-free translation. Prolactin mRNA sequences increased from 1.1 to 4.5% of the cytoplasmic poly(A)containing mRNA within 48 hr after addition of TRH to the medium; in parallel, growth hormone mRNA sequences were decreased. Similar effects on prolactin mRNA levels were observed in cells grown in serum-containing medium or when the cells were maintained in a serum-free medium for 72 hr prior to addition of TRH, suggesting that TRH is responsible for the stimulation of prolactin mRNA levels rather than for exerting a permissive effect for another factor present in serum.