The differentiation of three anterior pituitary cell types is regulated by the tissue-specific POU domain factor Pit-1, which is initially expressed on Embryonic Day 13.5–14 in mice. ThePit-1gene remains continuously, highly expressed in the somatotrope, thyrotrope, and lactotrope cells of the adult. Using the Pit-1-defective Snell dwarf as a genetic background, we demonstrate that thePit-1gene utilizes distinct enhancers for initial gene activation and for subsequent autoregulation (required for maintenance of expression) and that Pit-1-dependent activation of the distal enhancer can be mediated in the absence of the early enhancer. These two distinct enhancers provide the basis for temporally specific regulation by discrete pituitary-specific factors, events likely to be prototypic for regulation of other classes of genes encoding transcription factors controlling terminal differentiation.