Most cells are exquisitely responsive to calcium (Ca2+) (1) and hydrogen (H+) ions (i.e. pH) (2). In cardiac myocytes, Ca2+ ions trigger contraction and control growth and development (3), whereas H+ ions, which are generated or consumed metabolically, are potent modulators of virtually all biological processes (4). By acting on Ca2+-handling proteins directly or via other molecules, H+ ions exert both inhibitory and excitatory effects on Ca2+ signaling. Due to slow diffusion and common buffering, changes in cytoplasmic [Ca2+] or [H+] ([Ca2+]i, [H+]i) can become compartmentalized, leading potentially to complex spatial Ca2+/H+ coupling. We investigated the relationship between intracellular pH (pHi) and diastolic or resting [Ca2+]i by fluorescence-imaging of rat ventricular cardiac myocytes loaded with reporter-dyes for Ca2+ (Fluo3) or pH (cSNARF1). An increase in [H+]i, produced by superfusion of 80mM acetate (salt of membrane-permeant weak acid), evoked a 47±2% [Ca2+]i-rise, independent of sarcolemmal Ca2+-influx (replacing extracellular Na+ with N-methyl-D-glucamine and Ca2+ with EGTA), electrical pacing (2Hz field stimulation) or release from mitochondria (10 µM ruthenium-360), sarcoplasmic reticulum (10 µM thapsigargin or 10 mM caffeine) or acidic-stores (5 µM bafilomycin or 100 µM glycyl-L-phenylalanine 2-naphthylamide). Photolytic H+-uncaging from the membrane-permeant donor 2-nitrobenzaldehyde also raised [Ca2+]i. The [Ca2+]i-rise evoked by H+-uncaging or superfusion with acetate was absent in cells that were loaded with the pH-insensitive exogenous Ca2+-buffer BAPTA, either as the membrane-permeant acetoxymethyl ester or as the membrane-impermeant salt via patch-pipette (confirming that the H+-evoked [Ca2+]-rise is cytoplasmic). Inhibition of mitochondrial respiration with FCCP (1-5 µM), rotenone (10 µM), myxothiazole (10 µM), or of glycolysis with deoxyglucose (2 mM), reduced the H+-evoked [Ca2+]i-rise in a manner that correlated with the decline of [ATP] (determined by luciferase assay). H+-uncaging into buffer-mixtures in-vitro demonstrated that Ca2+-unloading from proteins, histidyl-dipeptides (HDPs; e.g. carnosine) and ATP can underlie the H+-evoked [Ca2+]i-rise. Raising [H+]i tonically at one end of a myocyte (regional exposure to 80 mM acetate using dual-microperfusion) evoked a local [Ca2+]i-rise in the acidic microdomain, which did not dissipate spatially but, instead, was maintained for as long as [H+]i was compartmentalised. Activating membrane-bound acid-extruding and acid-loading transporter-proteins on either end of the cell using dual microperfusion also produced standing pHi and [Ca2+]i gradient. These results are consistent with uphill Ca2+-ion transport into the acidic-zone via Ca2+/H+-exchange on diffusible HDPs and ATP molecules, energized by the spatial [H+]i-gradient. Ca2+-recruitment to a localized acid-microdomain was greatly reduced during intracellular Mg2+-overload (superfusion with 30 mM Mg2+ in the absence of Na+ and Ca2+) or by metabolic inhibition (rotenone and deoxyglucose), maneuvers that reduce the Ca2+-carrying capacity of HDPs (displacement of buffer-bound Ca2+ by Mg2+) and deplete ATP-levels. By exchanging Ca2+ for H+, diffusible cytoplasmic Ca2+/H+ buffer-molecules act like local ‘pumps’, producing uphill Ca2+-movement within cytoplasm, in response to H+-ion gradients. We conclude that cytoplasmic HDPs and ATP underlie spatial Ca2+/H+ coupling in the cardiac myocyte, by providing ion exchange and transport on common buffer-sites. Cytoplasmic histidyl-dipeptides and ATP thus act like a biological ‘pump’ without a membrane. Moreover, the 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. Given the abundance of cellular HDPs and ATP, spatial Ca2+/H+ coupling is likely to be of general importance in cell signaling.