The synthetic glucocorticoid, dexamethasone, stimulated the transepithelial electrical resistance and suppressed the DNA synthesis of 31EG4 nontransformed mouse mammary epithelial cells. The addition of transforming growth factor-beta 1 (TGF-beta) to mammary cells simultaneously with or up to 24 h after dexamethasone treatment prevented the steroid induction of transepithelial electrical resistance and stimulated the incorporation of [3H]thymidine. However, the TGF-beta inhibition of tight junction formation did not require de novo DNA synthesis. Confocal microscopy revealed that the organized immunostaining pattern of the tight junction protein, ZO-1, and F-actin at the cell periphery was disrupted by TGF-beta, resulting in disorganized and diffuse staining patterns throughout the cell. Western blot analysis demonstrated that TGF-beta did not alter the protein levels of ZO-1. In contrast to cells not treated or pretreated with steroid for up to 24 h, TGF-beta had no effect on cells pretreated with dexamethasone for 48 h. Transfection of chimeric reporter genes containing promoters responsive to either glucocorticoid or TGF-beta demonstrated that the mutual antagonism of tight junction dynamics by dexamethasone and TGF-beta occurs in the presence of intact signaling pathways. Taken together, our results establish for the first time that glucocorticoids and TGF-beta can antagonistically regulate tight junction formation in a nontransformed mammary cell line.