The principal acid extruding proteins in ventricular myocytes are Na+/H+ exchange (NHE1) and Na+-HCO3- cotransport (NBC, possibly comprising isoforms e1, e2 and n1) [1]. Although NHE1 and cardiac NBCs are trafficked to the surface membrane, their final spatial location has not been ascertained. We used protein immunofluorescence, pHi epifluorescence and confocal microscopy to explore transporter expression in the adult rat ventricular myocyte. Data are mean±SE, unpaired t-test was used, P<0.05 was considered significant. Monoclonal antibodies against NHE1 (BD Biosciences, Chemicon) and polyclonals against NBCe1, e2 or n1 (Chemicon and gifts from Dr J Praetorius, Aarhus University), all raised against cytoplasmic-tail epitopes, were tested. Western blots on protein from enzymically isolated ventricular myocytes identified NHE1, NBCe1, e2 and n1. To explore transporter distribution, fixed and permeabilised myocytes were incubated with the antibodies coupled to anti-rabbit or anti-mouse Alexafluor®488 and confocally imaged (n=4 to 60). NHE1 showed strong expression in the intercalated disc regions, with some occasional punctate surface distribution elsewhere. NBCe1 was strongly expressed in the transverse tubules (t-tubules), whereas e2 and n1 were more diffuse. Results suggest that NHE1 is selectively expressed in surface sarcolemma, principally towards the ends of myocytes, while NBC is also expressed in t-tubules. We next compared pHi regulation in control and detubulated myocytes. Detubulation was achieved using transient exposure to 1.5M formamide [2], and was confirmed by imaging cells loaded with the membrane dye, 8-di-ANEPPS (t-tubule staining was largely absent). As expected, detubulated cells displayed a reduced membrane capacitance (138±11 pF; n=10 vs 236±25 pF; n=14, P<0.05) and reduced L-type Ca2+-current amplitude (-2.5±0.5 pA/pF; n=9 vs -5.0±0.8 pA/pF; n=8, P<0.05) under whole cell patch clamp; and a spatially non-uniform upstroke of electrically-evoked Ca2+ transient (intracellular fluo-3), consistent with removal of a functional t-system. H+-equivalent efflux on NBC was measured during whole-cell pHi-recovery from an acid load (induced by NH4Cl prepulse in 5%CO2/22mMHCO3- Tyrode containing 30μM cariporide to block NHE; flux=dpHi/dt x intracellular buffering power). In detubulated cells, flux was reduced by 32-42% over the pHi range 6.55-6.85 (n=10, P<0.05). In contrast, H+ efflux on NHE (Hepes-buffered Tyrode) was unaffected (n=9, n.s.). We conclude that NHE and NBC transporters show differential spatial expression in the ventricular myocyte. As a result, acid extrusion via NHE occurs at surface sarcolemma and intercalated disc regions, while much acid extrusion through NBC takes place in the t-tubules. The two transporters may thus guard pHi in different cytoplasmic microdomains of the myocyte.