A specific and sensitive immunoprecipitation method for measurements of biosynthesized radioactive prolactin and growth hormone is described. Antisera to rat prolactin and growth hormone were developed in the rabbit and monkey, respectively. The specificity of the immune sera was assessed by polyacylamide gel electrophoresis of the dissolved immunoprecipitates. The two antisera showed cross-reactions with the nonhomologous hormone of less than 1%. Separation of tritium-labelled prolactin and growth hormone by immunoprecipitation, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was shown to be 95-57% complete. When both hormones were measured in the same microsample by sequential immunoprecipitation, the reaction was 97% complete for determination of intra- and extracellular prolactin and extracellular growth hormone, but 85% complete for determination of intracellular growth hormone. This method has been used to characterize the basal synthesis and secretion of prolactin and growth hormone in three different but related, pituitary cell strains. Radioactive prolactin and growth hormone was obtained from monolayer cultures when the cells were grown in the presence of [3H]L-leucine. The rate of prolactin synthesis and extracellular accumulation was higher than that of growth hormone in a cell strain which produced both hormones. In these cells prolactin synthesis represents 1-5%, and growth hormone 0.1-0.6% of total protein synthesis.