Transient sex differences of 5-hydroxytryptamine (5HT, serotonin) concentrations were found in the combined forebrain and midbrain of the neonatal rat. 1) Levels of 5HT in the female brain were significantly higher than those in the male on postnatal days 10, 12, and 14, though not on days 2, 4, and 8 or on days 16 and 25. 2) Castration of males on day 1 resulted in a nonsignificant elevation of brain 5HT on day 12 compared to intact males. 3) Ovariectomy of females on day 1 resulted in a significant reduction of brain 5HT on day 12 compared to intact females. 4) Injection of androgens in females on day 1 resulted in reduced brain 5HT measured on day 12 and day 14. 5) Injections of estrogens on day 1 elevated brain 5HT measured on days 8, 12, and 14 in females, and measured on days 8 and 12 in males. 6) When administered on day 11, these hormones had other effects: estrogen did not increase male brain serotonin measured on day 14, but did increase 5HT levels in the female brain; testosterone did not lower brain serotonin in either sex. 7) Given on day 20, estrogen did not alter 5HT levels, measured on day 25, in either sex. These effects of gonadal hormones on brain 5HT levels suggest an involvement of the gonads in maturation of the serotonin system of the brain and are discussed in relation to sexual differentiation of the brain. (Endocrinology93: 1329, 1973)