The influence of a partially purified extract of sheep hypothalami with in vivo growth hormone (GH) releasing activity was examined with respect to growth hormone synthesis and release in vitro. Rat anterior pituitaries were preincubated in the presence of 14C-leucine and then incubated in the presence of 3H-leucine and the hypothalamic extract. Effects on GH synthesis and on the release of both newly synthesized and stored GH were determined using radioimmunoassay and immunoprecipitation techniques. The hypothalamic extract increased release of newly synthesized GH while not affecting release of older, stored GH. This effect was distinguished from the simultaneous release of newly synthesized and stored GH caused by NH4+ and K+. Inhibition of GH synthesis occurred at high extract concentrations, but could be attributed to the toxic effects of the vehicle containing the extract. The consistent effects observed on the release of 14C- and 3H-labeled GH were not paralleled by those on immunoassayable GH release. The latter was influenced by many variables in vitro and proved an unreliable measure of release phenomena unless correlated with a simultaneous measure of cellular viability. The findings suggest the presence of two pools of GH in the pituitary which respond differentially to release stimuli and which are distinguishable according to time of synthesis. The effect of the hypothalamic extract appears to be limited to the newly synthesized hormone pool.