To reinvestigate the "hydrolase-related transport" concept, neutral -D-glucosidase, a membrane-bound disaccharidase of renal proximal tubule, was first purified from brush-border membranes and then asymmetrically reincorporated into egg phosphatidylcholine vesicles. Proteolytic treatments and immunotitration studies demonstrated that this enzyme was integrated in native and artificial membrane vesicles with a similar topology. The uptake of free glucose and glucose produced by maltose hydrolysis was studied using 1) proteoliposomes containing integrated neutral -D-glucosidase, in combination with other membrane proteins, and 2) proteoliposomes containing only the other membrane proteins but incubated in a medium containing neutral -D-glucosidase in its hydrophilic form. No modification was observed in the uptake of free D-glucose or D-glucose produced by maltose hydrolysis, regardless of enzyme localization. In contrast to previous findings on the hydrolase-related transport concept, these results rule out any participation of neutral -D-glucosidase in the transport of free glucose or glucose produced by maltose hydrolysis. Hydrolytic activity and transmembrane transport appear to be two independent and sequential steps.