Folliculostellate (FS) cells are known to communicate with each other and with endocrine cells via gap-junctions in the anterior pituitary. We investigated whether transforming growth factor 3 (TGF-3) and estradiol, known to regulate FS cell production and secretion of basic fibroblast growth factor (bFGF), increases gap-junctional communication to alter bFGF secretion from FS cells. FS cells in monolayer cultures were treated with TGF-3 or vehicle alone for 24 h and then microinjected with Lucifer Yellow and high molecular weight Texas Red dextran. Ten minutes later the transfer of dye among adjacent cells was recorded with a digital microscope. TGF-3 increased the transfer of dye. The TGF-3 neutralizing antibody and the gap-junction inhibitor octanol reduced the effect of TGF-3 on the transfer of dye. The TGF-3-induced transfer of dye was unaltered by simultaneous treatment with estradiol. The steroid alone also had no effect. TGF-3 increased total and phosphorylated levels of connexin 43 (Cx43). Estradiol treatment did not produce any significant changes on basal or TGF-3-induced increases in Cx43 levels. The gap-junction inhibitor octanol reduced TGF-3-increased levels of bFGF in FS cells. Taken together, these results suggest that TGF-3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion.