An in vitro superfusion system was used to examine the effects of cycloheximide on the responsiveness of hemipituitaries from male Rana pipiens chronically treated with gonadotropin-releasing hormone (GnRH). Control hemipituitaries superfused with medium (DME) alone showed a rapid initial response to 100 ng/ml GnRH, and LH (luteinizing hormone) and FSH (follicle-stimulating hormone) levels remained above baseline throughout up to 13 h of treatment with GnRH. Subsequent to a 2-h rinse with DME alone following the initial treatment with GnRH, these control tissues showed a highly augmented increase in gonadotropin secretion in response to a final hour of GnRH superfusion, suggesting self-priming with as little as 5 h of initial GnRH treatment. Hemipituitaries treated with 71 microM cycloheximide showed a similar rapid initial response to 100 ng/ml GnRH, but levels declined to less than in control tissues within 3 h. Moreover, these tissues exhibited only slight responses when challenged with GnRH a second time. There were also no significant differences in response to GnRH by glands pretreated with cycloheximide for either 3 or 9 h. These results indicate the presence of a rapidly releasable pool of gonadotropin responsible for the initial response to GnRH that has minimal requirements for protein synthesis, and a second pool dependent upon protein synthesis that is involved in the maintenance of elevated gonadotropin secretion in response to chronically superfused GnRH, and for self-priming.