A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845-1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogue o-nitrophenyl-beta-D-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products.