Cytoplasmic Ca2+ is modulated by H+-ions. We investigated the relationship between intracellular pH (pHi) and diastolic or resting Ca2+ ([Ca2+]dia) in confocally-imaged rat myocytes loaded with Fluo-3 (Ca2+) or cSNARF-1 (pHi). Intracellular release of H+-ions from a membrane-permeant weak-acid (80mM acetate; pHi decreases from 7.2 to 6.6) produced a 0.6±0.03µM (mean±SEM) rise in [Ca2+]dia (N>50). Intracellular H+-release from a photo-labile caged H+-compound (2-nitrobenzaldehyde; 0.5-2mM) also produced a rise in [Ca2+]dia. The rise in [Ca2+]dia was restricted spatially to the acid-microdomain formed by H+-release (N>50). The resulting cytoplasmic Ca2+-gradient was maintained for the lifetime of the underlying pHi-microdomain, but could be dissipated by AM-loaded BAPTA (100µM; a Ca2+-buffer; N=20). The spatial H+-Ca2+ interaction was not abolished by removal of extracellular Na+ or Ca2+, or by exposure to thapsigargin (10µM), caffeine (10mM), bafilomycin (5µM), glycyl-L-phenylalanine 2-naphthylamide (100µM) or ruthenium-360 (10µM) (N=10-50). However, inhibition of mitochondrial respiration with FCCP (1-5µM), rotenone (10µM), myxothiazole (10µM), or of glycolysis with deoxyglucose (2mM) (N=10-20), reduced the H+-evoked [Ca2+]dia-rise in a manner that correlated with the decline of [ATP] measured by luciferase assay (pHi-[Ca2+]dia relationships shifted left by 0.3 pHi units as [ATP]i is halved). We conclude that the H+-evoked Ca2+-rise is not due to extracellular Ca2+-influx, or Ca2+-release from organelles. It most likely arises from competitive Ca2+/H+-binding to cytoplasmic buffers, some of which are diffusible and therefore engage in spatial Ca2+/H+ exchange, resulting in recruitment of Ca2+ to acidic microdomains. We therefore investigated the in vitro pH-sensitivity of Ca2+-binding to a range of known cytoplasmic buffers, using H+-uncaging in agar-set mixtures. We find that histidyl-dipeptides (e.g. carnosine) and ATP are the myocyte’s principal pH-sensitive mobile Ca2+-buffers (physiological 7.5 mM ATP and 10 mM carnosine release 45±2µM and 30±3µM µM Ca2+ per pH unit decrease, respectively, N=9). Involvement of Ca2+-bound ATP confers metabolic sensitivity to the cytoplasmic H+-Ca2+ interaction. Results indicate that cytoplasmic pHi-microdomains, formed during membrane H+- or weak-acid transport will generate Ca2+-microdomains. These may help to compensate for the reactive effects of H+-ions on Ca2+-dependent protein function.