Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.