The basal rate of pre-labeled stored 14C-labeled rat growth hormone ([14C]rGH) release from perifused rat pituitary explants is a constant fraction of pituitary GH content, suggesting random release from the storage pool. Basal release of newly synthesized [3H]rGH occurs in two phases: 1) immediate and associated with [3H]rGH synthesis, and 2) late (delayed by 60 min) and independent of concurrent [3H]rGH synthesis. Dibutyryl cyclic AMP (10(-2)M)-stimulated release of stored [14C]rGH is characterized by an initial acute rise followed by a second phase of continuous rapid release. Immediate and late release of new [3H]rGH is increased by dibutyryl cyclic AMP, and the late phase of [3H]rGH is less delayed. Simultaneous exposure of pituitary explants to [3H]alanine and [14C]leucine resulted in the release of immunoprecipitable rGH whose ratio of incorporated 3H and 14C varied with time. The observed changes suggest that after it is synthesized, a GH molecule may either be released directly or be processed into the somatotroph's storage compartment. In addition, stored GH is composed of two pools, one of which is immediately releasable. The differential incorporation of [3H]alanine and [14C]leucine into "big" and "small" rGH, together with the ability to differentially displace 3H-labeled "big" and 14C-labeled "small" rGH from the GH antibody suggest that "big" rGH is a heterogenous molecule including "small" rGH and another peptide rather than simply a dimer of "small" rGH.