The Zebrafish is a tropical teleost fish that has been the focus of increasing numbers of developmental studies. Physiological interest in this species has been spurred by the ease of mutants can be induced by chemomutagenesis. However, our understanding of the basic physiology of Zebrafish, in particular cardiac excitation-contraction coupling is limited. Indeed, there is currently no information about the electrical activity of single myocyte freshly isolated from Zebrafish ventricle heart; this study addressed this point. Heart was removed post mortem from Zebrafish and ventricle was then minced and stir gently in isolating solution containing trypsin and collagenase for 30 min. The extracellular physiological solution was (in mM): 150 NaCl, 5.4 KCl, 1.5 MgSO4, 0.4 NaH2PO4, 2 CaCl2, 10 glucose, 10 HEPES (pH 7.7 with NaOH). The whole cell configuration of the patch clamp was used to record Na current (INa), Ca current (ICa) and action potential (AP). Patch pipettes were filled with internal solution (in mM): 139 KCl, 10 NaCl, 1 MgCl2, 20 HEPES, 5 MgATP, 0.3 NaGTP (pH 7.2 with KOH). Cell capacitance was determined using the membrane test protocol of Clampex. During voltage clamp experiments, holding potential was -80 mV and stimulation frequency was 0.1 or 1 Hz. Results are presented as mean±SE and analyzed with paired t-test. Single ventricular myocytes from Zebrafish are long and thin, as described for other fish species. Cell capacitance was 27.4±2.5 pF (n=17). ICa density (test pulse to 0 mV) was -11.2±2.9 pA/pF and the time to reach 37% of ICa peak was 29.2±2.6 ms (n=7). INa density (test pulse to -40 mV) was -104±22 pA/pF (n=5). Increasing stimulation frequency to 1 Hz decreased peak INa by 10±5% and increased peak ICa by 20±7% (p0.05). However, increasing stimulation frequency to 2 Hz significantly decreased AP overshoot (n=6, p<0.05) and reduced dV/dt (n=6, p=0.06). To conclude, we have developed a method to obtain viable isolated ventricle myocytes from Zebrafish heart. Ionic currents studied present characteristics similar to other fish species. The presence of a plateau during the AP suggests that this species might be appropriate for ion channels related mutation screening of cardiac alteration.