In cardiac myocytes, the opening of voltage-activated sodium (Na_V) channels induces a fast-activating inward Na⁺ current which mediates the rapid action potential upstroke. The slowly inactivating (or late) component of the Na⁺ current (I_LNa) remains active during prolonged depolarisation and contributes to the action potential plateau. Lysophosphatidylcholine (LPC) accumulates in the ischaemic myocardium and is arrhythmogenic, at least in part via effects on I_LNa. Here, we examined the mechanisms underlying the effects of LPC on I_LNa, using recombinant Na_V1.5 α subunits expressed in HEK293 cells. Na⁺ currents were studied by whole-cell patch-clamp. Currents were evoked by 40ms step depolarisations to -40mV (holding potential -140mV) every 5s. Data were taken from current values obtained 10ms after initiating the depolarisation. Accordant with previous reports, 5μM LPC enhanced I_LNa. Thus, normalised (I/I₀) I_LNa was 2.53±0.58 (n=7) after a 6min exposure to LPC, compared to 0.99±0.13 (n=8) in cells perfused without LPC (P<0.05). Enhancement of I_LNa was abolished by the antioxidant ascorbic acid (200μM) and by the NADPH oxidase inhibitor DPI (5μM). Normalised I_LNa following LPC exposure in the presence of these agents was 1.21±0.08 (n=7) and 1.16±0.16 (n=5) respectively (P<0.05 compared to LPC alone in each case). Although these data suggest a role for NADPH oxidase derived superoxide in mediating the response to LPC, the superoxide donor pyrogallol (100μM) did not affect I_LNa. Normalised I_LNa following exposure to pyrogallol was 1.15±0.05 (n=8; P>0.05 compared to control). Enhancement of I_LNa was also abrogated when cells were pretreated with the NOS inhibitors L-NAME (500µM) and 7-nitroindazole (300µM). Normalised I_LNa following LPC exposure in the presence of these agents was 0.88±0.06 (n=6) and 1.37±0.14 (n=6) respectively (P<0.05 in each case compared to LPC alone). Despite this, the NO donor SNAP (200μM) evoked no change in I_LNa. Normalised I_LNa following exposure to SNAP was 1.11±0.10 (n=6; P>0.05 compared to control). However, SNAP and pyrogallol combined enhanced I_LNa, to 2.16±0.35 (n=8; P<0.05 compared to control), suggesting peroxynitrite formation as a mediator of the response to LPC. Supporting this, the peroxynitrite scavenger FeTPPS (50µM) abolished the response to LPC (normalised I_LNa in response to LPC in the presence of FeTPPS was 1.15±0.11, n=4; P<0.05 compared to LPC alone). We conclude that peroxynitrite formation provides a novel mechanism by which LPC regulates Na⁺ channel function.