Mechanisms of cardiac pacemaking and conduction system (CPCS) development are difficult to study, due in part to the absence of models that are physiologically similar to humans in which we can label the entire CPCS. Investigations of the adult rabbit heart have provided insight to normal and abnormal cardiac conduction. The adult and embryonic rabbit has an endogenous marker of the entire cardiac conduction system, neurofilament 160 (NF-160). Previous work suggested that ventricular septation correlates with critical phases in avian CPCS development, in contrast to the mouse CPCS. Combining high resolution optical mapping with immunohistochemical analysis of the embryonic rabbit heart, we investigated the significance of ventricular septation in patterning the rabbit embryonic conduction system. We hypothesized that completion of ventricular septation does not correlate with changes in the ventricular activation sequence in rabbit embryos, and that CPCS anatomy determines the activation sequence of the embryonic heart. We found that pre-septated (day 11-13, n=13) and post-septated hearts (day 15, n=5) both had similar "apex-to-base" ventricular excitation. PR intervals were not significantly different in either group. CPCS anatomy revealed continuity of the NF-160+ tract connecting the presumptive sinoatrial (SA) node, atrioventricular (AV) junction, and ventricular conduction system. The presence of collagen in the AV junction coincided with the appearance of an AV interval. Conclusions: The "apex-to-base" ventricular activation sequence in the rabbit embryo is present before completion of ventricular septation. CPCS anatomy reflects global cardiac activation as demonstrated by high resolution optical mapping.