We have developed a sensitive homologous radioimmunoassay for mouse prolactin (mPRL) capable of measuring concentrations of PRL as low as 1 ng/ml of mouse serum and studied PRL secretion in mice under several physiological conditions. Newborn mice of C3H/St strain had low concentration of PRL in sera at 2–5 days of age which increased with age. Adult females of C3H/St strain contained significantly more PRL in their pituitary glands (7.9 μg/mg) than comparable females of C57BL/St strain (5.6 μg/mg); in contrast, however, they had lower PRL levels in sera (26.6 ng/ml) than the C57BL/ St females (84.5 ng/ml). The females of both strains possessed greater concentration of PRL in their pituitary glands, but in sera only C57BL/St females showed higher levels than males. Castration of C3H mice reduced pituitary PRL in both sexes but decreased serum PRL in males only. Stilbestrol in the diet of castrated mice, however, enhanced pituitary and serum PRL levels in either sex. Adult C3H mice had depressed serum concentrations of PRL following the injection of L-dopa and elevated levels after epinephrine. (Endocrinology91: 1045, 1972)