The rhythmic contraction of the vertebrate heart is dependent on organized propagation of electrical excitation through the cardiac conduction system. Because both muscleand neuron-specific genes are co-expressed in cells forming myocardial conduction tissues, two origins, myogenic and neural, have been suggested for this specialized tissue. Using replication-defective retroviruses, encoding recombinant β-galactosidase (β-gal), we have analyzed cell lineage for Purkinje fibers (i.e., the peripheral elements of the conduction system) in the chick heart. Functioning myocyte progenitors were virally tagged at embryonic day 3 of incubation (E3). Clonal β-gal⁺ populations of cells, derived from myocytes infected at E3 were examined at 14 (E14) and 18 (E18) days of embryonic incubation. Here, we report that a subset of clonally related myocytes differentiates into conductile Purkinje fibers, invariably in close spatial association with forming coronary arterial blood vessels. These β-gal⁺ myogenic clones, containing both working myocytes and Purkinje fibers, did not incorporate cells contributing to tissues of the central conduction system (e.g. atrioventricular ring and bundles). In quantitative analyses, we found that whereas the number of β-gal⁺ myocyte nuclei per clone more than doubled between E14 and E18, the number of β-gal⁺ Purkinje fiber nuclei remained constant. These data provide evidence that: (1) Purkinje fibers and working myocytes share a common myogenic precursor in the embryonic tubular heart; (2) differentiation of Purkinje fibers may involve earlier commitment to non-proliferation than that of working myocytes and; (3) the peripheral (i.e., the intramural Purkinje fiber network) and central components of the cardiac conduction system are derived from independent parental cells, and that these two components are linked together to establish the sequentially integrated conduction system of the adult heart. We discuss potential mechanisms for induction of Purkinje fibers from embryonic myocytes in association with coronary vasculogenesis.