Multiple related cis-active elements required for cell-specific activation of the rat prolactin gene appear to bind a pituitary-specific positive transcription factor(s), referred to as Pit-1. DNA complementary to Pit-1 mRNA, cloned on the basis of specific binding to AT-rich cell-specific elements in the rat prolactin and growth hormone genes, encodes a 33 kd protein with significant similarity at its carboxyl terminus to the homeodomains encoded by Drosophila developmental genes. Pit-1 mRNA is expressed exclusively in the anterior pituitary gland in both somatotroph and lactotroph cell types, which produce growth hormone and prolactin, respectively. Pit-1 expression in heterologous cells (HeLa) selectively activates prolactin and growth hormone fusion gene expression, suggesting that Pit-1 is sufficient to confer a characteristic pituitary phenotype. The structure of Pit-1 and its recognition elements suggests that metazoan tissue phenotype is controlled by a family of transcription factors that bind to related cis-active elements and contain several highly conserved domains.