Tissue-specific transcription is advantageously investigated by using viral promoters, which are selected for compact regulatory elements. Mouse mammary tumor virus (MMTV) has adapted to specialized cell types and targets initially B lymphocytes. We previously showed that, in B-cell lines, glucocorticoid-induced MMTV transcription requires an ETS family factor, GA-binding protein (GABP), bound in tandem to the MMTV DNA next to the glucocorticoid receptor (GR). We now report that transforming growth factor beta (TGF-beta) superinduces this response up to 10-fold through binding of its effectors, Smads, between the GABP-binding motifs. The basal level was unaffected. The TGF-beta-glucocorticoid cooperation also depended on GR and GABP binding, was transferable to another promoter, and occurred both with transiently transfected and with integrated templates. Smad3 associated in vitro with GR, with GABPalpha (via the MH2 domain), and with GABPbeta, Smad4 only with GABPalpha. Interactions of Smad3 with GABP (when coexpressed or endogenous to B cells) were shown by coprecipitation and by mammalian two-hybrid assay. This composite DNA element integrates three signaling pathways deriving from TGF-beta, glucocorticoid hormones, and a unique ETS factor, and may allow MMTV to exploit factors from the milk. It may as well indicate novel possibilities for cellular regulatory networks.