The cscB gene encoding a putative sucrose permease from Escherichia coli EC3132 [Bockmann, J., Heuel, H. & Lengeler, J.W. (1992) Mol. Gen. Genet. 235, 22-32] was placed under control of the lacZ promoter/operator and overexpressed. Cells of E. coli K-12 expressing cscB gene product from a high copy number plasmid accumulate sucrose against a concentration gradient with an apparent Km of about 1 mM and a Vmax of approximately 23 nmol/min/mg protein. In contrast, transport of lactose or melibiose is not observed. Epitope-tagging and immunological analysis reveal that concomitant with increased transport activity, a protein with an apparent M(r) of 33-34 kDa appears in the cytoplasmic membrane. The results provide direct support for the contention that the cscB gene encodes a sucrose permease and provide the basis for further molecular analysis.