Membrane-bound, flavin-linked D-lactate dehydrogenase in membrane vesicles of E. coli ML 308-225 is solubilized by extraction with guanidine HCl. When membrane vesicles prepared from a D-lactate dehydrogenase mutant are treated with this extract, they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent transport. Similar effects are obtained with wild-type membrane vesicles in which D-lactate oxidation and D-lactate-dependent transport have been inactivated by 2-hydroxy-3-butynoate. Although treatment of wild-type vesicles with the extract results in an increased capacity to catalyze D-lactate oxidation, no effect on transport is observed. Reconstituted transport activity is a saturable function of the amount of guanidine extract added. Moreover, the quantity of extract required to achieve maximum initial rates of transport varies with each transport system. On the other hand, reconstituted D-lactate oxidation increases linearly over a broader range of extract concentrations.Oxamate, a competitive inhibitor of D-lactate dehydrogenase, and p-chloromercuribenzenesulfonate block both the initial rate of transport and the steady-state level of accumulation in reconstituted vesicles. Furthermore, these reagents induce efflux of transport substrates from preloaded, reconstituted vesicles. The same reagents inhibit the initial rate of uptake but not the steady-state level of accumulation in ML 308-225 vesicles, and do not induce efflux. These results suggest that, although reconstituted vesicles catalyze D-lactate oxidation and D-lactate-dependent transport, the system has not been reconstituted to its native state.