In the present study, we used a three-dimensional pituitary cell culture system from early postnatal rats to examine the in vitro developmental potential of triiodothyronine (T3) and thyrotrophin-releasing hormone (TRH). Cell types were identified at the hormone mRNA level by single-cell reverse transcription-polymerase chain reaction and any change in abundance was further examined by immunofluorescence staining of the corresponding hormone protein. In aggregates from 14-day-old rats, long-term (12-16 days) treatment with T3 (0.5 nM) increased the abundance of cells expressing prolactin mRNA (PRLmRNA cells) by 2.5-fold and lowered that of nonhormonal cells and thyroid-stimulating hormone beta (TSHbeta)mRNA cells. The abundance of growth hormone (GH)mRNA cells decreased during culture compared to that in the freshly dispersed pituitary gland and T3 did not significantly affect this cell population. Cells coexpressing PRL mRNA and GH mRNA virtually disappeared during culture but reappeared in the presence of T3. T3 increased the abundance of PRL-immunoreactive (ir) cells in aggregates from 14-day-old rats, as well as in aggregates from newborn and 1-week-old rats. As estimated by bromodeoxyuridine (BrdU) labelling, a 3-day treatment with T3 enhanced the number of PRL-ir cells that had incorporated BrdU, but did not yet expand the total population of PRL-ir cells. Long-term treatment with TRH (100 nM) did not affect the proportion of PRLmRNA or GHmRNA cells, but consistently increased the proportional number of TSHbeta(mRNA) and TSHbeta-ir cells. The present data confirm the findings obtained in recent in vivo loss of function genetic studies suggesting that T3 plays a prominent role in postnatal expansion of the lactotroph population and that TRH is important for thyrotroph development. The data suggest that the effect of T3 is brought about by a direct action on the pituitary gland through a cell proliferation mechanism. T3 also appears to support the lactosomatotroph population. In view of the established theory that lactotrophs develop from GH-expressing progenitor cells and that this is a post mitotic event, we propose that T3 is mitogenic for GHmRNA cells that lack GH-ir material and that transdifferentiate into PRL-ir cells, but that a pathway of PRL cell development from mitotic nonhormonal cell progenitors may also be involved.