Intracellular acidosis is a feature of both myocardial ischaemia, where it has been linked to ischaemia-reperfusion injury1, and heart failure2. A decrease in pHi has been shown to have multiple effects on the calcium handling apparatus3, leading to the hypothesis that acidosis may drive myocardial pathology via effects on calcium. Here, we show that altering pHi globally and locally affects multiple parameters of spontaneous Ca2+ waves, which may be an arrhythmogenic risk factor. pHi was altered globally by superfusing with 20-80 mM acetate (acidosis) or 20 mM trimethylamine (alkalosis). Spontaneous Ca2+ waves, induced by raising superfusate Ca2+, were linescan-imaged at 37°C in rat isolated ventricular myocytes AM-loaded with fluo-3. Parallel pHi measurements were made using AM-loaded SNARF-1. Data are mean±SE, P values calculated using a paired Student’s t test. Intracellular acidosis increased spontaneous wave frequency up to 360±62% of control (P=0.0002, n=18). This effect was prevented by inhibiting the sarcolemmal Na+/H+ exchanger (NHE) with 30 μM 5-(N,N-dimethyl)amiloride, demonstrating Na+-coupling to intracellular Ca2+ dynamics. Under these conditions, waves were suppressed relative to control (to 61±22% of control; P=0.0023, n=11), indicating that H+ may have cardioprotective properties. Intracellular alkalosis also decreased wave frequency to 60% of control after 60 seconds (P=0.0048, n=10). Decreasing pHi increased wave velocity up to 140±3% of control (P<0.0001, n=17), while an intracellular alkalosis decreased velocity to 80% of control (P<0.0001, n=12). Analysis over a range of pHi values demonstrated a linear relationship between pHi and wave velocity. Computational modelling4 suggests that this may be due to a decrease in cytoplasmic Ca2+ buffering; consistent with this, inhibiting NHE had no further significant effect during acidosis. It is likely that pHi in the ischaemic heart is heterogeneous, therefore to investigate whether differences in pHi affect local Ca2+ properties, we induced a stable pHi gradient (pHi 6.6-7.3) in single ventricular myocytes by superfusing two parallel microstreams perpendicular to the cell, one containing normal Tyrode, the other 80 mM acetate5. With NHE inhibited, wave initiation was inhibited in the acidic microdomain (31±7% of control;P<0.05, n=13) and stimulated in the more alkaline microdomain (164±25%; P<0.05, n=13), while wave velocity was increased in the acidic microdomain and decreased in the more alkaline microdomain. Other effects on Ca2+ dynamics, including wave decay, also mapped on to the pHi gradient. We conclude that pHi locally regulates Ca2+ handling and dynamics, and thus pHi heterogeneity may provide a substrate for aberrant Ca2+ signalling in cardiac pathology.