The zebrafish (ZF) has emerged as an important model for developmental cardiovascular (CV) biology, however, much less is known about the cardiac function of the adult zebrafish that can be used as a model of teleost CV biology. Here, we describe electrophysiological parameters, such as HR, AP duration (APD) and atrio-ventricular (AV) delay, in the ZF heart over a range of physiological temperatures (18-28 °C). Using the potentiometric dye RH-237 the electrical activity in several distinct regions of the heart were assessed simultaneously. While intrinsic HR varied considerably between fish, the ex vivo preparation exhibited impressively stable HRs and sinus rhythm for >5 hours, with a mean HR of 158 ± 9 bpm (mean ± SEM) (n = 20) at 28 °C. Atrial and ventricular APDs at 50% repolarization (APD₅₀) were 33 ± 1 ms and 98 ± 2 ms, respectively. The AV delay was 61 ± 3 ms at 28 oC. APD and AV delay did not appear to be statistically dependent on intrinsic basal HR, likely due to the innate beat-to-beat variability within each heart. Cooling to 18 °C decreased HR by ~40% and atrial and ventricular APD₅₀ increased by ~3x and 2x, respectively. The acute effect of temperature was most apparent on the atrial APD. In conclusion, optical mapping studies utilizing zebrafish heart can yield insightful information into cardiac arrhythmias as well as serving as a model for other teleosts.