Pyruvate is a key metabolic intermediate and the substrate for diacetyl and acetoin synthesis. The mechanism of pyruvate transport was determined in Lactobacillus plantarum by use of cells and membrane vesicles. In the cells, protonophores inhibited pyruvate transport, whereas valinomycin did not. Pyruvate was accumulated against a gradient in membrane vesicles. The transport rate and the degree of accumulation increased as the proton gradient increased, but an imposed K potential of -61 mV did not drive pyruvate transport. The maximum transport rate (35 nmol/min/mg protein) and accumulation ratio (162-fold) were at pH 3.0, with an apparent Km value of 35-mu-M. These results suggested that pyruvate was transported by a proton symport.