Dispersed anterior pituitary cells from adult female rats were separated by gradient sedimentation at unit gravity. The small-sized cell population on top of the gradient consisted of 65.6 ± (SE) 4.2% (n = 8) cells immunoreactive to antiserum against S-100 protein, a marker of folliculo-stellate (FS) cells in rat pituitary. The corresponding fraction derived from adult male or immature female rats were also enriched in S-100 positive cells but to a lower extent. Only small numbers of S-100 positive cells were found in medium- and large-sized cell populations. Coaggregating the S-100 cell-enriched populations from adult females with other pituitary cell populations resulted in a clear-cut inhibition of the GH response to rat GHreleasing factor and /3-adrenergic agents, of the PRL response to TRH and angiotensin II (All) and the LH response to LHRH. The magnitude of inhibition increased with the number of FS cells put into the coaggregates. In perifused aggregates prepared from different gradient fractions from immature females, there was a negative correlation between the occurrence of FS cells and the magnitude of the PRL response to All. The low responsiveness to All in FS cell enriched aggregates was not abolished when these aggregates were redissociated into single cells. It is suggested that FS cells constitute an intercellular messenger system for local inhibitory control of pituitary hormone secretion which is not based on direct and intimate contact between the interacting cells. (Endocrinology120: 685–691, 1987)