pdx1 (pancreatic and duodenal homeobox gene-1), which is expressed broadly in the embryonic pancreas and, later, in a more restricted manner in the mature β cells in the islets of Langerhans, is essential both for organ formation and β cell gene expression and function. We carried out a transgenic reporter gene analysis to identify region- and cell type-specific regulatory regions in pdx1. A 14.5-kb pdx1 genomic fragment corrected the glucose intolerance of pdx1^+/− animals but, moreover, fully rescued the severe gut and pancreas defects in pdx1^−/− embryos. Sequences sufficient to direct reporter expression to the entire endogenous pdx1 expression domain lie within 4.3 kb of 5′ flanking DNA. In this region, we identified two distinct fragments that drive reporter gene expression to different sets of islet neuroendocrine cells. One shows pan-endocrine cell specificity, the other is selectively activated in insulin-producing β cells. The endocrine-specific regulatory regions overlap a localized region of 5′ flanking DNA that is remarkably conserved in sequence between vertebrate pdx1 genes, and which has been associated with β cell-selective expression in cultured cell lines. This region contains potential binding sites for several transcription factors implicated in endodermal development and the pathogenesis of some forms of type-2 diabetes. These results are consistent with our previous proposal that conserved upstream pdx1 sequences exert control over pdx1 during embryonic organogenesis and islet endocrine cell differentiation. We propose that mutations affecting the expression and/or activity of transcription factors operating via these sequences may predispose towards diabetes, at least in part by direct effects on endocrine pdx1 expression.