The rhythmic contraction of the vertebrate heart is dependent on organized propagation of electrical excitation through the cardiac conduction system. Because both muscle- and 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 beta-galactosidase (beta-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 beta-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 beta-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 beta-gal+ myocyte nuclei per clone more than doubled between E14 and E18, the number of beta-gal+ Purkinje fiber nuclei remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)