An enriched population of cells immunoreactive to antiserum against S-100 protein, a marker of folliculo-stellate (FS) cells in the rat pituitary, was obtained by separation of dispersed pituitary cells from adult female rats by gradient sedimentation at unit gravity. The effect of FS cells on the stimulation and inhibition of prolactin (PRL) and growth hormone (GH) release was studied by coaggregation experiments of the FS cell-enriched population with respectively a lactotroph-enriched and a somatotroph-enriched population from adult female rats. The FS cell population not only attenuated the stimulation of PRL and GH release, but also significantly attenuated the inhibition of PRL release by 10, 30 or 300 nM dopamine (DA), and the inhibition of GH release by 0.1 nM somatostatin (SRIF). The stimulatory action of angiotensin II (AII) on PRL secretion in the presence of DA was also attenuated by the FS cells. Light microscopic evaluation of immunostained semithin sections showed a meshwork of cytoplasmic extensions of FS cells as well as follicular structures in the aggregates. There was no preferential association of FS cells with certain cell types. The permeability of the aggregates to diffusing molecules was tested at the ultrastructural level by the lanthanum hydroxide tracing technique. Lanthanum traced the intercellular gaps over the entire aggregate irrespective of whether the proportional number of FS cells was high or low, indicating that FS cells do not seal off certain areas in the aggregate by the formation of tight junctions. It is suggested that FS cells attenuate the action not only of stimulatory but also inhibitory secretagogues on hormone-secreting pituitary cells. The possible physiological relevance of the present findings is supported by the topological distribution of the FS cells in the aggregates, which closely resembles that of the intact pituitary.