The prolactin (PRL) response of perifused rat pituitary tissue to the cholinomimetic agent carbachol (CARB) was studied under various in vitro conditions. Perifusion of freshly removed hemipituitaries from 14-day-old rats with CARB did not affect basal PRL release. When established in organ culture for 3 days in a serum-free chemically defined medium, there was a significant increase of PRL release in response to CARB. This PRL releasing activity of CARB depended on the hormonal environment of the culture medium: supplementation of the culture medium with triiodothyronine (T3) and the glucocorticoid dexamethasone (DEX) completely reversed the PRL releasing activity of CARB into an inhibition of PRL release. In dispersed pituitary cells from immature rats, cultured as three-dimensional reaggregates, a similar reciprocal responsiveness to CARB existed which was also determined by T3 and DEX. This reciprocal responsiveness to CARB was preserved in adult female rats but was shifted to a more prominent inhibition in adult male rats. Tumoral PRL secreting GH3 cells, cultured as aggregates, always responded in an inhibitory way, irrespective of the hormonal environment. The expression of the reciprocal responses, in particular of the inhibitory pathway to CARB was dependent on close cellular contacts, as the inhibitory response of normal and tumoral pituitary cells, cultured as isolated cells on Cytodex beads, was completely absent. The stimulatory response of normal lactotrophs, cultured as isolated cells was, although attenuated, still preserved. The present data suggest that there exists a reciprocal responsiveness of normal lactotrophs to cholinomimetics depending on the hormonal environment and close cellular associations. In contrast, only inhibitory PRL responses occur in GH3 tumoral lactotrophs, which are not dependent on thyroid and glucocorticoid hormones.