A detailed description of the electrophysiological features of cardiomyocytes in the various contractile chambers of the vertebrate heart is essential to understand the evolution of cardiac electrical activity, yet very little is known about reptiles. The present study characterizes major ionic currents (I _Na, I _CaL, I _Kr, I _K1 and I _KACh) and action potential (AP) configuration in cardiomyocytes from the ventricle, the right atrium and the sinus venosus (SV) of Burmese pythons (Python molurus) using sharp microelectrode and patch clamp recordings. Special attention was given to SV, since it consists of myocardial cells and appears to contribute to right atrial filling in snakes. We demonstrate that most of the SV in pythons has a stable resting potential of − 82.3 ± 2.6 mV (n = 9) and lacks pacemaker activity. AP duration at 50% repolarization was similar in cells from SV and atria (350.2 ± 8.7 and 330.4 ± 17.2 ms, respectively; n = 7), but shorter than ventricular APs (557.6 ± 19.2 ms, n = 5) at 30 °C. The densities of ionic currents, however, differed substantially between atrial and SV cells, where the latter had much lower densities of I _Na, I _CaL and I _Kr than atrial and ventricular myocytes. I _K1 in ventricle was ninefold greater than in atrial cells and 23-fold greater than in myocytes from SV. However, I _KACh was absent in ventricular cells, while it was equally large in atrial and SV myocytes. Consistent with this observation, APs of atrium and SV, but not ventricle, were greatly shortened upon addition of acetylcholine (10 ⁻⁶ M). Thus, snake SV, right atrium and ventricle have distinct patterns of ionic currents, but the resulting electrical activity is similar in atrium and SV.