Pituitary cells from adult male rats subjected to chronic (6 and 10 weeks) medial hypothalamic ablation (MHA) were analyzed by unit gravity sedimentation to assess distribution of size and density of lactotrophs, and for subsequent in vitro prolactin (PRL) release in primary culture. Tinctorial staining (Herlant's tetrachrome) showed that initial preparations of cells from MHA rats were small and relatively undifferentiated. MHA cells did not sediment as far into the gradient as did cells from intact control pituitaries. Intracellular PRL content was lower in all gradient fractions of MHA cells. At 6 weeks after surgery, peak recovery of PRL was also in the upper portions of the gradient. In the 10-weeks group, however, peak PRL recovery from MHA cells was in a population that sedimented further, but more restrictedly, in comparison with control cells. At both postsurgical intervals, the majority of tinctorially or immunocytochemically identified lactotrophs from lesioned rats were lower in the gradient, indicating enlarged and denser cells. Relative numbers of lactotrophs (per pituitary) were increased 10 weeks after MHA. In vitro PRL release, over a maximum of 21 days culture, was comparable for cells from MHA rats and intact controls, according to daily per cell secretion rates and "production index" (hormone released/initial hormone content). By comparison, luteinizing hormone (LH) release was suppressed in culture compared to intact controls, and LH was recovered from gradient fractions of smaller cells. The results indicate that chronic removal of hypothalamic influence results in gradual prolactin cell hypertrophy and decreased hormone retention and in relative increase in numbers.(ABSTRACT TRUNCATED AT 250 WORDS)