Two members of the POU-domain family of transcriptional activators, Pit-1 and Oct-1, are coexpressed in cells of the anterior pituitary gland. We demonstrate that the pituitary-specific developmental regulator Pit-1 can bind as a heterodimer with the widely expressed 100-kD Oct-1 protein to critical tissue-specific cis-active elements in the rat prolactin gene. Pit-1 and Oct-1 can also associate, in the absence of DNA, via their POU domains. Coexpression of Pit-1 and Oct-1 in a heterologous cell type results in reproducible 2- to 2.5-fold synergistic transcriptional effects on genes under control of the native prolactin promoter or of a single Pit-1 response element. These results suggest that a combinatorial pattern of heterodimeric and homodimeric interactions between different members of the POU-domain gene family can result when members of this large family are coexpressed in cells of developing and established organ systems, potentially regulating differential developmental gene activation.