Isolated pituitary cells from 14-day-old female, 14-day-old male, and adult male rats were separated into several fractions by unit gravity sedimentation. With preparations from 14-day-old animals, the percentage of basophils increased from top to bottom of the gradient. The bottom fractions contained large basophils with a purity attaining almost 90% in the female and 75% in the male preparation. The same fraction prepared from adult male rats was composed of over 80% of large acidophils. As identified by immunostaining, the majority of the female large basophils were gonadotrophs, whereas in 14-day-old males about 1/3 of these basophils were thyrotrophs. The relative proportion of medium and small sized gonadotrophs and of thyrotrophs was also higher in the 14-day-old rats than in the adults. An estimate of the distribution of FSH and LH among the gonadotrophs was made by comparing the number of cells reacting with anti-FSH, with anti-LH, and with a mixture of both antisera. The data suggested that irrespective of cell size, the majority of the gonadotrophs in 14-day-old rats contained both FSH and LH. In most fractions, the occurrence of cells with only one hormone was more apparent in the males than in the females. In adult rats, cells with only FSH were most abundant in the fraction with large gonadotrophs. The distribution of hormone content, as measured by RIA, was highly characteristic for each animal group. In 14-day-old females, more than 70% of the recovered hormones was in the large gonadotrophs. In 14-day-old and adult males, substantial amounts of hormone were also recovered from medium sized gonadotrophs. The present findings suggest a relationship between developmental changes in pituitary hormone secretion and changes in pituitary cell type distribution.