At an unknown point in mammalian development, cardiac precursor cells become committed to the cardiocyte phenotype. Certain of these are believed to specialize further into pacemaker cardiocytes. By culturing explanted embryonic ventricles into in vivo organ culture (Tucker DC, Snider C, Woods WT Jr: Pediatr Res 23:637-642, 1988), we observed pacemaker cells arising apparently from cardiocytes. We hypothesized that this event can be triggered by intercellular attachments, innervation, vascularization, or other factors. The present study was designed to test the hypothesis that primitive ventricular cells in the tubular heart can organize into an anatomically and electrophysiologically distinct pacemaker structure in the absence of innervation or vascularization from extrinsic sources. Developing ventricles of tubular hearts from 10-d-old rat embryos (n = 22) were excised and incubated in culture dishes. Within each explant, a group of In the developing mammalian heart, cells committed to the cardiocyte phenotype appear early, and they are initially quiescent (1, 2). At a later unknown point in development, some of these cells acquire the pacemaker phenotype. The sequence of steps between quiescent cardiocyte and spontaneously beating cardiocyte are poorly understood. This laboratory reported that 12-d-old rat ventricles explanted and cultured in oculo were quiescent in culture for 4 ? 1 d. Then spontaneous beating began, and a pacemaker locus was observed at the point of contact between cardiac tissue and the host iris (3). Growth of blood vessels, nerves, extracellular matrix, or iris tissue from the host into the explants was postulated as potential stimuli for organization of the new pacemaker. Pacemaker cells in the new center resembled those of the immature mammalian sinus node and atrioventricular node (4), suggesting that they might be precursors of the cardiac conduction system.In the present study, influences of extrinsic blood vessels, nerves, and iris tissue were eliminated by explanting embryonic rat ventricles into culture dishes instead of anterior eye Received November 30, 1993; accepted November 14, 1994