To examine the substrate specificity of the membrane transport carriers LldP(L-lactate permease) and GlcA (glycolate permease) of Escherichia coli, a mutant strain lacking their structural genes and blocked in the metabolism of the tested substrates was constructed and transformed with a plasmid bearing either the lldP or the glcA gene. Each transformant acquired the ability to accumulate L-lactate, D-lactate, and glycolate against a high concentration gradient. Substrate accumulation was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force. Competition Of C-14-L-lactate transport by nonradioactive L-lactate, D-lactate, and glycolate in LldP synthesizing cells and competition of C-14-glycolate transport by the same three substrates in GlcA synthesizing cells showed that both carriers effectively transported all three substrates with a K-i value ranging from 10 to 20 muM. D-Lactate does not appear to have a permease of its own. Utilization of the compound depends mainly on LldP. (C) 2002 Elsevier Science.