Acidosis, as found in myocardial ischaemia, potentially triggers arrhythmogenic Ca2+ signalling1, with clinical implications. Here we characterise the effect of reducing pHi on Ca2+ waves in rat isolated ventricular myocytes. Ca2+ waves (induced by raising Ca2+o from 1 to 5 or 7.5mM) were imaged confocally with AM-loaded fluo-3 (linescan, 37°C). Parallel measurements of pHi were made in cSNARF-1 AM-loaded cells. pHi was reduced (up to 0.8 units) by superfusion of 80mM acetate. Results are mean±SEM, P values calculated using a paired Student’s t test unless otherwise stated. Reducing pHi first suppressed (16±6% of control after 20s; P<0.0001, n=18) then increased (360±62% after 60s; P=0.0002, n=18) wave frequency. Inhibiting the sarcolemmal Na+/H+ exchanger (NHE; 30μM 5-(N,N-dimethyl)amiloride2) attenuated the secondary increase, as after 60s frequency recovered to only 61±22% (P=0.0023; n=11). An increase in wave velocity after 60s was seen with (140±3%; P<0.0001, n=17) and without (126±6%; P=0.002, n=10) NHE activity. These data agree with a previous report3. A decrease in pHi for ≥60s slowed wave relaxation (time constant τcontrol=87±3ms, τacetate=97±2ms; P=0.02, one-way ANOVA, n=6). Local effects of pHi on Ca2+ waves were studied by inducing a spatial pHi gradient in a myocyte (by microperfusing two parallel solution streams perpendicular to the cell using a double-barrelled glass micropipette4). One half of the cell was superfused with normal Tyrode, the other with 80mM acetate (both containing 7.5mM Ca2+). This produced a stable longitudinal pHi gradient (pHi 6.6 to 7.3). With NHE inhibited, a decrease in wave frequency (31±7% of control; P<0.05, n=13) was seen in the acidic microdomain and an increase (164±25%; P<0.05, n=13) in the more alkaline microdomain. Conversely, wave velocity was increased (118±5% of control; P<0.05, n=6) and decreased (85±4%; P<0.05, n=6) in acidic and alkaline microdomains respectively. Thus, wave properties map onto a spatial gradient of pHi, indicating that pH-dependent control of Ca2+ waves is a locally regulated phenomenon. Computational modelling of Ca2+ waves5 suggests that a slowing of wave decay may reflect SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) inhibition. Although the acute effect of SERCA inhibition is to increase wave velocity (by extending the diffusibility of cytoplasmic Ca2+), at steady-state, however, a decrease in wave velocity is predicted due to a fall in sarcoplasmic reticulum Ca2+ content (a factor that affects ryanodine receptor opening probability). This suggests that other H+-targeted sites may be involved in the observed increase. We conclude that spatial pHi heterogeneity elicits multiple localised effects on Ca2+ signalling, suggesting that it may contribute to the heterogeneity of Ca2+ signalling observed in myocardial ischaemia.