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Synthesis of prolactin was measured in a clonal strain of functional pituitary cells in culture by incubating the cells with [3H]leucine; labeled prolactin was separated from the other labeled proteins by specific immunoprecipitation followed by dissociation with sodium dodecyl sulfate and disc gel electrophoresis. Accumulation of labeled prolactin in the cells was linear for at least 1 hour; labeled prolactin was released into the culture medium after 1 hour. Treatment with 28 nmthyrotropin-releasing hormone for 3 days increased prolactin accumulation in the medium 2.5-fold, and the increased synthesis of prolactin completely accounted for the increased accumulation. Treatment with 5 x10-6mhydrocortisone for 7 days decreased prolactin accumulation in the medium 2- to 5-fold, and the decrease in synthesis completely accounted for the decreased accumulation. Therefore the long term (3 to 7 days) effects on these cells of thyrotropinreleasing hormone and hydrocortisone result only in a change in the synthesis of prolactin and not in its rate of degradation.