Although the critical role of the His-Purkinje system (HPS) in the propagation of cardiac action potentials from the atria to the ventricular myocardium in the mature heart is well appreciated, its functional and anatomical development are not well understood. The embryonic heart begins beating early in development devoid of a mature conduction system, and the HPS cannot be identified by conventional histochemical analysis until the seventh embryonic day of chicken development. Although many biochemical markers have been found that apparently identify HPS precursors, little is known about how these biochemical markers function in the maturation of the cardiac conduction system. Using immunohistological techniques, we demonstrated that the maturation of the HPS may be observed by the expression of two distinct populations of conduction system precursors, identified by the expression of cell surface carbohydrates PSA-NCAM (PSA) and HNK-1, both of which are known to participate in cell-cell and cell-substrate interactions in the development of the nervous system. By stage 25, PSA was detected in ventricular trabeculae and the interventricular septum (IVS) in a pattern that resembles bundle branches and Purkinje fibers. Beginning at stage 28, HNK-1 epitope expression in the IVS was observed in myocardium just superior to the PSA-positive bundles in a pattern that resembles the common His bundle. This junctional region was also positive for atrial myosin heavy chain (aMHC), another marker for the HPS. These data suggest that a complex, coordinated process of biochemical and morphological change governs the development of the cardiac conduction system.