We report the cloning, chromosomal localization, and analysis of the expression pattern of epicardin, a member of the basic helix-loop-helix (bHLH) family of transcription factors. Within its bHLH domain, the human and murine epicardin genes were most similar to paraxis, a bHLH gene important for segmentation of embryonic paraxial mesoderm. In situ hybridization studies revealed strong epicardin expression in murine embryos at 9.5 days postcoitum (dpc) in a region of the septum transversum at the base of the heart known as the proepicardial organ. This mesenchymal structure extends villous projections from which epicardial precursor cells emerge and migrate out over the surface of the myocardium. Strong expression was seen in individual migratory cells and clusters at 9.5 dpc and in a continuous epicardial cell layer in more mature hearts. Also from 9.5 dpc, epicardin transcripts were seen in endocardial cushions of the atrioventricular canal and outflow tract, in skeletal myoblasts within branchial arches and in condensing mesenchyme of gut, kidney, urinary tract, gonads, spleen, and lung. Northern analysis showed that expression persisted in mature visceral organs and heart, but was transient in skeletal muscle. The central role played by bHLH factors in pathways for tissue determination in the embryo suggests a function for epicardin in specification of select mesodermal cell populations associated with heart, cranial skeletal muscle, gut, and urogenital system.