Intracellular acidosis and pro-arrhythmogenic Ca2+ waves are features of myocardial ischaemia. Intracellular acidosis stimulates Ca2+ waves via sarcolemmal Na+/H+ exchange (NHE) activity raising Na+i, and hence Ca2+i, via Na+/Ca2+ exchange. But when NHE is inhibited, intracellular acidosis suppresses waves, demonstrating that H+ has both stimulatory and inhibitory effects1. Intracellular pH becomes spatially non-uniform during NHE activity, and heterogeneity of myocardial pHi occurs during regional ischaemia2. We therefore investigated the effect of a spatial pHi gradient on Ca2+ waves in rat isolated ventricular myocytes subjected to modest Ca2+ overload (5 mM Ca2+o, to induce spontaneous waves). Data are given as mean±SEM, P values calculated by paired t test.A longitudinal pHi gradient (6.6 to 7.3) was imposed using a dual microstream apparatus (80 mM acetate superfused over one end of a myocyte). Spontaneous waves were inhibited in the acidic end (frequency reduced to 25±17% of control, P = 0.0071, n = 6) but strongly stimulated in the distal, non-acidic end (five-fold increase, P = 0.022, n = 6). When NHE activity was blocked (30 μM 5-N,N-dimethylamiloride: DMA), wave suppression persisted in the acidic end, but stimulation in the non-acidic end was now only two-fold (n = 7). Thus, locally enhancing sarcolemmal NHE activity triggers downstream Ca2+ waves. As the pHi gradient was kept constant by the dual microperfusion, this Ca2+ signalling must have been secondary to Na+ influx into the acidic zone. As reported previously3, this will drive rapid Na+ diffusion to less acidic regions, where Ca2+ waves can be triggered.The possible involvement of additional SR-based mechanisms was tested by quantifying the effect of the pHi gradient on local Ca2+ spark frequency. This frequency declined by 72±11% (P = 0.0002, n = 9) in the acidic end, consistent with ryanodine receptor (RyR) inhibition by H+. In support of this, superfusing one end of a myocyte with the RyR antagonist tetracaine (tet, 0.5 mM) recapitulated the effect on Ca2+ waves of local acidosis plus DMA i.e. it inhibited waves in the tet-exposed region, with only a small (two-fold) increase downstream (n = 12). Thus, wave suppression in acidic zones is due to direct H+ blockade of RyRs, but most of the downstream stimulation of Ca2+ waves is due to upstream NHE stimulation, with Na+i acting as a mobile, cytoplasmic messenger. A pHi gradient thus provides a substrate for the localized triggering of Ca2+ waves. This mechanism may help to explain the border zone clustering of Ca2+ waves during regional myocardial ischaemia, given that ischaemic zones are acidic and intracellular Na+ ions will readily diffuse downstream through gap junctions.