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An extract of ovine hypothalami, partially purified on Sephadex, inhibited the synthesis and release of GH in vitroby rat anterior pituitaries incubated sequentially in the presence of 14C-leucine and then 3H-leucine with the inhibitory hypothalamic factor (GIF). Effects on GH synthesis and on the release of both newly synthesized and stored GH were determined using an immunoprecipitation technique. GIF decreased GH synthesis and decreased release of newly synthesized GH while not affecting release of older, stored GH. This effect contrasted with that of a hypothalamic GH releasing factor (GRF) which increased the release of newly synthesized GH while not affecting the rate of GH synthesis or the release of older GH. GIF inhibition was reversible since glands preincubated in its presence later responded characteristically to GRF. GIF reduced the release of both newly synthesized and stored GH produced by 21mM K. Prolactin synthesis and release were not affected by GIF or GRF. The findings support the concept of two pools of pituitary GH. Each pool, distinguishable according to time of synthesis, was differentially affected by GIF and GRF with both hypothalamic factors exerting primary effects upon the newly synthesized pool.