Abstract Previous studies using neonatal rat cardiomyocytes have reported antiarrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs). In this study, we examined the effects of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on the spontaneous contractile activity and membrane fluidity of adult rat ventricular myocytes. Cardiomyocytes were induced to contract spontaneously by continuous superfusion of a solution containing the arrhythmogenic agents isoproterenol (a β-adrenergic receptor agonist) or lysophosphatidylcholine. The percentage of cardiomyocytes displaying spontaneous contractions induced by isoproterenol when pretreated with the saturated fatty acid docosanoic acid was 48.1 ± 7.7%; the percentage for cardiomyocytes pretreated with DHA was 7.1 ± 2.4% (P < 0.01). DHA significantly prevented lysophosphatidylcholine-induced spontaneous contractions (17.7 ± 6.5%) compared with treatment with the saturated fatty acid stearic acid (78.0 ± 7.3%, P < 0.01). The membrane fluidizing agent benzyl alcohol also significantly prevented spontaneous contractions in cardiomyocytes. Membrane fluidity was determined by steady-state fluorescence anisotropy (r ss) using the fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl) trimethyl-ammonium p-toluene-sulfonate (TMAP-DPH). DHA and benzyl alcohol dose-dependently decreased the r ss; however, saturated fatty acids were without effect. These results suggest that the antiarrhythmic mechanisms of the n-3 PUFAs such as DHA may involve changes in membrane fluidity.