The anti-arrhythmic actions of omega-3 polyunsaturated fatty acids (PUFAs) are widely documented, although the mechanism of this protection is not well understood. The primary objective was to investigate the effects of long chain omega-3 PUFAs on refractoriness, conduction velocity, and defibrillation on a healthy heart in vivo. We infused 0.77ml/kg i.v. Omegaven (EPA + DHA, n=10), Intralipid (vehicle control, n=10), or saline (n=10) over 20 minutes to anesthetised New Zealand White rabbits. Pre and post drug/vehicle treatment, we measured ventricular effective refractory period (VERP: Cycle Length=180, 160, 140msec), shortest cycle length allowing 1:1 capture (SCL), right ventricular monophasic action potential duration at 90% repolarization (MAPD90), QRS duration (stimulus to end of QRS), and QT interval (measured during ventricular pacing). Acute omega-3 PUFA administration has small but significant class III effect in vivo, decreases defibrillation threshold, and may also increase conduction velocity. This combination of effects could possibly account for their anti-arrhythmic actions.