Isolated pituitary cells from 14-day-old male and female and adult male rats were separated into enriched populations of gonadotrophs by velocity sedimentation at unit gravity. The cells, collected from five gradient fractions, were established in monolayer culture. Hormone content and hormonal secretion in response to LHRH were measured after 3 days in culture. Stored FSH and LH declined during culture, but the relative differences between fractions and between animal groups were maintained. In all fractions, LHRH stimulated the release of both FSH and LH in a dose-response fashion. The secretory potential per gonadotroph changed with fraction and the pattern was highly characteristic for each animal group. In 14-day-old females, both FSH and LH secretion increased 20 times from the fraction with the smallest to the fraction with the largest gonadotrophs. In preparations from 14-day-old males, only the release of FSH consistently rose with gonadotroph cell size. With cells from adult males, FSH as well as LH release increased with cell size, but the pattern was different for each hormone. Secretion did not always correlate with the cellular content of hormone before stimulation. The present findings suggest that gonadotrophs isolated from animals in a different physiological or developmental status may retain in short term culture certain of their previous functional characteristics. There is evidence that the gonadotroph cell population is heterogeneous not only in terms of the magnitude of the response to LHRH but also in terms of the relative proportion of FSH and LH secreted. Changes in the relative proportion of certain variants of gonadotrophs may be a cellular basis for differential regulation of FSH and LH secretion by a single releasing hormone.