Recently, a cDNA (pRS1) was cloned from pig kidney cortex that encodes a membrane-associated protein involved in Na(+)-coupled sugar transport. pRS1 alters sugar transport by SGLT1 from rabbit intestine or by SMIT from dog kidney which is homologous to SGLT1. In contrast, pRS1 does not influence transporters from other genetic families. We report the cloning of the intronless human gene hRS1 (6,743 bp), which encodes a 617-amino-acid protein with 74% amino acid identity to pRS1. By fluorescence in situ hybridization, hRS1 was localized to chromosome 1p36.1. The localization to one chromosome and Southern blot analysis of restricted genomic DNA suggest that there is only one RS1-homologous gene in humans. Functionality of hRS1 was demonstrated by co-expression experiments of hRS1 and SGLT1 from human intestine in oocytes from Xenopus laevis. They show that hRS1-protein inhibits Na(+)-D-glucose co-transport expressed by human SGLT1 by decreasing both the Vmax and the apparent Km value of the transporter. The analysis of the 5'-noncoding sequence of hRS1 revealed different enhancer consensus sequences that are absent in the SGLT1 gene, e.g., several consensus sequences for steroid-binding proteins.