The pro-opiomelanocortin (POMC) gene is expressed very early during pituitary development, before expression of the other pituitary hormone genes, growth hormone and prolactin, and before expression of the Pit-1/GHF-1 transcription factor which activates those genes. Thus, analysis of the POMC promoter should provide markers of the early stages of pituitary development at the time when cells are being committed to expression of one or the other pituitary hormone. We have previously localized the rat POMC promoter to a 543-bp 5′-flanking DNA fragment of the gene using transfection and transgenic mice experiments. We have now used mutagenesis and in vitro protein-DNA binding studies to define three domains of the promoter which have distinct and complementary activities. Within these domains which require each other for full activity, at least nine regulatory elements were defined by in vitro footprinting and replacement mutagenesis. Each element appeared equally important for promoter activity, as mutagenesis of any element had similar effect on promoter activity. Most of the elements bound different AtT-20 nuclear proteins in gel mobility shift experiments. Whereas only two elements appeared to be binding sites for the known transcription factors AP-1 and chicken ovalbumin upstream promoter, the seven other elements appeared to bind nuclear proteins with novel properties. Thus, in contrast to the predominant role of Pit-1/GHF-1 in transcription of the growth hormone and prolactin genes, the control of an early pituitary gene, POMC, appears to depend on the synergistic interaction of several regulatory elements which bind different nuclear proteins.