Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.