It is generally accepted that under basal conditions there is preferential release of newly synthesized hormone by a number of endocrine cell type, including those that secrete GH or PRL. However, the cellular basis for this phenomenon along with the relative contribution of stored hormone to basal secretion has yet to be clearly established. In the present study, we employed reverse hemolytic plaque assays to monitor basal and stimulated release of GH and PRL from individual cells in which de novo protein synthesis had been blocked. Monodispersed pituitaries from adult male rats were cultured for 21 h in the absence or presence of maximally effective doses of puromycin (100 microM) or cycloheximide (36 microM) and were then subjected to separate plaque assays for GH or PRL. Treatment with puromycin reduced the percentage of GH or PRL secretors (plaque formers) by about half. Coincubation with stimulatory secretagogues did not increase the percentages of GH or PRL secretors in control cultures, but returned the proportion in puromycin-treated cells to normal, demonstrating that cells which failed to secrete basally could still release hormone from their stored pools when stimulated. Very similar results were obtained when these experiments were repeated with cycloheximide. Taken together, these results demonstrate that only a fraction of the cells that release GH or PRL are dependent upon newly synthesized hormone for basal secretion; the remainder appear capable of mobilizing stored hormone for this purpose even in the absence of stimulation.