Folliculostellate (FS) cells are known to communicate with each other and with endocrine cells via gap junctions in the anterior pituitary. We investigated whether TGFbeta3 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 TGFbeta3 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. TGFbeta3 increased the transfer of dye. The TGFbeta3-neutralizing antibody and the gap junction inhibitor octanol reduced the effect of TGFbeta3 on the transfer of dye. The TGFbeta3-induced transfer of dye was unaltered by simultaneous treatment with estradiol. The steroid alone also had no effect. TGFbeta3 increased total and phosphorylated levels of connexin 43. Estradiol treatment did not produce any significant changes on basal or TGFbeta3-induced increases in connexin 43 levels. The gap-junction inhibitor octanol reduced TGFbeta3-increased levels of bFGF in FS cells. Taken together, these results suggest that TGFbeta3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion.