Background: There are many reports about the anti-arrhythmic effects of omega-3 polyunsaturated fatty acids, however, the mechanisms are still not completely delineated. The purpose of this study was to investigate the characteristics of action potentials and transient outward potassium currents (I-to) of Sprague-Dawley rat ventricular myocytes and the effects of docosahexaenoic acid (DHA) on action potentials and I-to. Methods: The calcium-tolerant rat ventricular myocytes were isolated by enzyme digestion. Action potentials and I-to of epicardial, mid-cardial and endocardial ventricular myocytes were recorded by whole-cell patch clamp technique. Results: 1. Action potential durations (APDs) were prolonged from epicardial to endocardial ventricular myocytes (P < 0.05). 2. I-to current densities were decreased from epicardial to endocardial ventricular myocytes, which were 59.50 +/- 15.99 pA/pF, 29.15 +/- 5.53 pA/pF, and 12.29 +/- 3.62 pA/pF, respectively at + 70 mV test potential (P < 0.05). 3. APDs were gradually prolonged with the increase of DHA concentrations from 1 mu mol/L to 100 mu mol/L, however, APDs changes were not significant as DHA concentrations were in the range of 0 mu mol/L to 1 mu mol/L. 4. I-to currents were gradually reduced with the increase of DHA concentrations from 1 mu mol/L to 100 mu mol/L, and its half-inhibited concentration was 5.3 mu mol/L. The results showed that there were regional differences in the distribution of action potentials and I-to in rat epicardial, mid-cardial and endocardial ventricular myocytes. APDs were prolonged and I-to current densities were gradually reduced with the increase of DHA concentrations. Conclusion: The anti-arrhythmia mechanisms of DHA are complex, however, the effects of DHA on action potentials and I-to may be one of the important causes.