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The release of melanocyte-stimulating hormone (MSH) into the medium during incubation and the pituitary tissue content of MSH were measured separately using pituitary glands collected from rats at various stages of the oestrous cycle. The MSH was measured by a biological assay using a synthetic -MSH as standard. The release of MSH was maximal during the pro-oestrous phase and MSH content of the pituitary gland was highest during dioestrus. The influences of the tripeptide Pro-Leu-Gly-NH₂, which inhibits MSH secretion in vivo, and of progesterone on the release of MSH in vitro were studied with tissue collected at various phases of the oestrous cycle. Pro-Leu-Gly-NH₂ was effective in inhibiting MSH release both at pro-oestrus and oestrus but not at dioestrus. Progesterone overcame this inhibition.