1. The fall of intracellular pH (pH1) following the reduction of extracellular pH (pH0) was investigated in guinea-pig isolated ventricular myocytes using intracellular fluorescence measurements of carboxy-SNARF-1 (to monitor pH1). Cell superfusates were buffered either with a 5% CO2-HCO3- system or were nominally CO2-HCO3-free. 2. Reduction of pH0 from 7.4 to 6.4 reversibly reduced pH1 by about 0.4 pH units, independent of the buffer system used. 3. In HCO3(-)-free conditions, acid loading in low pH0 was not dependent on Na(+)-H+ exchange or on the presence of Na+. It was unaffected by high-K+ solution, by voltage-clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ and Ba2+) and by the organic Ca2+ channel blocker diltiazem, thus ruling out proton influx through H(+)-or Ca(2+)-conductance channels or influx via a K(+)-H+ exchanger. The fall also persisted in the presence of glycolytic inhibitors, or the lactate transport inhibitor, alpha-cyano-4-hydroxy cinnamate. 4. In HCO3(-)-free conditions, acid loading in low pH0 was reversibly inhibited (by up to 85%) by Cl(-)0 removal and was slowed by the stilbene drug DBDS (dibenzamidostilbene disulphonic acid). In contrast, the Cl(-)-HCO3-exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) had no inhibitory effect. Acid loading is therefore mediated by a novel Cl(-)-dependent, acid influx pathway. 5. After switching to CO2-HCO3(-)-buffered conditions, acid loading was doubled. It was still not inhibited by Na(+)-free or high-K+ solutions but was once again inhibited (by 78%) in Cl(-)-free solution. The HCO3(-)-stimulated fraction of acid loading was inhibited by DIDS. 6. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl(-)-HCO3-exchange, while we suggest the other to be a novel Cl(-)-OH-exchanger (although we do not rule out the alternative configuration of H(+)-Cl-co-influx). The proposed dual acid-loading mechanism accounts for most of the sensitivity of pH1 to a fall of pH0.