A pituitary cell population of 14-day-old female rats, containing lactotropes and somatotropes but deprived of gonadotropes, was prepared by unit gravity sedimentation through a serum albumin gradient and allowed to reaggregate either as such or after mixing this population with cells of the gonadotropic alpha T3-1 cell line. In a perifusion system gonadotropin-releasing hormone (GnRH) had no effect on prolactin (PRL) and growth hormone (GH) release in the former aggregates but stimulated PRL release in the latter. In the alpha T3-1 cell-containing aggregates GnRH showed a biphasic effect on GH release: inhibition during exposure to GnRH followed by a rebound secretion upon removal of the peptide. The aggregation capacity of alpha T3-1 cells with the normal pituitary cells was demonstrated by using an alpha T3-1 cell clone stably transfected with the reporter gene beta-galactosidase. Perifusion of the gonadotrope-deprived aggregates with medium conditioned by alpha T3-1 cell provoked a rapid stimulation of PRL release and a biphasic effect on GH release. Medium conditioned by the corticotropic cell line AtT20 also stimulated PRL release but had no concomitant effect on GH release. Medium conditioned by alpha T3-1 cells, when added for 40 h to aggregates of 14-day-old rat pituitary, provoked an increase in the number of 3H-thymidine (3H-T)-labelled lactotropes and a decreased in the number of 3H-T-labelled somatotropes. The conditioned medium was concentrated on Sep-Pak C18 and ultrafiltrated through an Amicon membrane with 3-kD molecular weight cut-off and the retained molecules separated by reversed-phase HPLC. The material stimulating 3H-T labelling of lactotropes eluted from the column with a different retention time than material inhibiting 3H-T labelling of somatotropes, suggesting that the effect on lactotropes is mediated by (a) molecule(s) different from that affecting somatotropes. The effects of alpha T3-1 cells and their secretion products on lactotropes and somatotropes were comparable to those we previously observed using enriched populations of normal gonadotropes. The HPLC elution profiles of the substances affecting 3H-T incorporation as well as the specificity of these effects were also similar to that of the substances isolated previously from gonadotrope-conditioned medium. The present data, therefore, support previous conclusions on the paracrine control of the lactotrope/somatotrope lineage by the gonadotropes. Further purification and chemical characterization of the growth factors with selective action on lactotropes and somatotropes may lead to a better understanding of the development of the latter lineage.