Major difficulties investigating the developing cardiac conduction system stem from that the embryonic heart is extremely small (< 2 mm) and cardiac activation is relatively rapid (< 8 msec). The objective of this study was to investigate the electrophysiology of the embryonic chick cardiac conduction system at periseptation stages with a photodiode array-based detection method of optical mapping capable of high spatial and temporal resolution. Previous work indicated that, in chicken embryos, a switch occurs in ventricular activation pattern from immature base-to-apex to mature apex-to-base pattern at the time of ventricular septation. It was our aim to map activation in more detail to identify the active pathway or pathways of atrioventricular conduction at these particular stages. Analysis of preseptated hearts (n = 10) showed that the latest atrial activation took place just above the site of the earliest ventricular activation at the ventral left ventricular base. Analysis of postseptated hearts (n = 11) showed apex-to-base conduction consistent with activation through the maturing His–Purkinje system. Evaluation of hearts during septation revealed a gradual transition of ventricular activation patterns rather than an abrupt “switch.” External pacing of preseptated hearts revealed significant slowing of interventricular conduction compared with spontaneous beats (spontaneous, 61.7 cm/sec ± 9 cm/sec vs. paced, 36.5 cm/sec ± 10 cm/sec). The more detailed mapping revealed that, before septation, the pattern of activation of the ventricular myocardium is consistent with direct atrial–ventricular myocardial connections at the left lateral atrioventricular junction; however, functional evidence for a preferential conduction pathway within the ventricles was present before septation. Developmental Dynamics 233:456–465, 2005. © 2005 Wiley-Liss, Inc.