Macrophages perform phagocytic and effector activities in a number of different tissues. The environment of the inflammatory foci in which they function is often acidic and contains an abundance of lactate. We characterized the ability of thioglycollate-elicited mouse peritoneal macrophages to accumulate lactate from the medium and to use this lactate to maintain intracellular energy stores. Lactate uptake was stereospecific for L-lactate and was inhibited by the organic anion transport blocker probenecid but not by concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid that block anion exchangers. L-[14C]Lactate uptake was not affected by variation of the extracellular Na+ concentration but was enhanced by acidification of the extracellular medium, suggesting that lactate uptake was mediated by a proton cotransport system. The enhanced accumulation of [14C]-lactate seen in medium at pH 6.0 to 6.5 was inhibited by probenecid or by an excess of unlabeled L-lactate. When macrophages were incubated in PBS without glucose for 6 h, intracellular stores of phosphocreatine were 13 nmol/mg of protein, compared with 44 nmol/mg of protein in cells incubated in medium containing glucose. When lactate was substituted for glucose, phosphocreatine stores were 32 nmol/mg of protein. These studies reveal that macrophages take up L-lactate in a pH-dependent manner and that lactate uptake occurs via a probenecid-inhibitable monocarboxylate transporter; they suggest that macrophages can utilize this lactate as an energy source.