Cardiac activity of the turtle (Trachemys scripta) is greatly depressed with cold acclimation and anoxia. We examined what electrophysiological modifications accompany and perhaps facilitate this depression of cardiac activity. Turtles were first acclimated to 21°C or 5°C and held under either normoxic or anoxic (6 h at 21°C; 14 d at 5°C) conditions. Then, we measured cardiac action potentials (APs) using spontaneously contracting whole-heart preparations and whole-cell current densities of sarcolemmal ion channels using isolated ventricular myocytes under appropriate normoxic and anoxic conditions. Compared with 21°C-acclimated turtles, 5°C-acclimated turtles exhibited a less negative resting membrane potential (by 18-29 mV), a 4.7- to 6.8-fold slower AP upstroke rate and a 4.2- to 4.9-fold greater AP duration (APD). Correspondingly, peak density of ventricular voltage-gated Na⁺ (I_Na) and L-type Ca²⁺ currents and inward slope conductance of inward rectifier K⁺ (I_K1) channel current were approximately 1/7th (Q10 = 3.4), 1/13th (Q10 = 5.0), and half that (Q10 = 1.4) of 21°C-acclimated ventricular myocytes, respectively. With anoxia at 21°C, peak I_Na density doubled and ventricular APD increased by 47%; a change proportional to the reported ~30% reduction of intrinsic heart rate. In contrast, with anoxia at 5°C, ventricular AP characteristics were unaffected, and of the ion currents investigated, only the inward conductance via I_K1 changed significantly (reduced by 46%). The present findings indicate that cold temperature, more so than prolonged anoxia, results in substantial modifications of cardiac APs and reduction of ventricular ion current densities. These changes likely prepare cardiac muscle for winter anoxia conditions.