This study examined the effects of dietary incorporation of n-3 polyunsaturated fatty acids (PUFAs) into cardiac membrane phospholipids on Ca(2+) handling (using Fura-2) and arrhythmic contractility in electrically-stimulated, adult rat ventricular cardiomyocytes. Dietary lipid supplementation with fish oil (FO) for 3 weeks significantly increased the proportion of total n-3 polyunsaturated fatty acids (in particular, docosahexaenoic acid) in ventricular membrane phospholipids compared with a saturated fat (SF) supplemented diet (26.2 +/- 0.9% vs 6.9 +/- 0.9%, respectively, P < 0.001). Cardiomyocytes isolated from the FO group were significantly (P < 0.001) less susceptible to isoproterenol-induced arrhythmic contractile activity compared with the SF group over a range of isoproterenol concentrations. Isoproterenol (0.5 &mgr;M) stimulation increased end-diastolic and systolic [Ca(2+)](i) to a similar extent in both groups. The time constant of Ca(2+) transient decay was significantly increased in the FO group compared with the SF group (98.4 +/- 2.8 ms, n = 8 and 86.9 +/- 2.1 ms, n = 8, P < 0.01, respectively). The effect of dietary n-3 PUFA incorporation into membrane phospholipids was not associated with changes in sarcoplasmic reticulum Ca(2+) content (measured by rapid application of caffeine) or membrane fluidity. The increase in the time constant of decay of Ca(2+) transients following dietary supplementation with FO may indicate altered functioning of the sarcolemmal Na(+)-Ca(2+) exchanger by n-3 PUFA incorporation into membrane phospholipids.