We describe in this report the cDNA structure, functional characteristics, genomic organization, and promoter analysis of the mouse H+-coupled low-affinity peptide transporter PEPT1. The mouse PEPT1 cDNA cloned from a kidney cDNA library is ∼3.1 kb long and encodes a protein of 709 amino acids. When expressed heterologously in mammalian cells and in Xenopus laevis oocytes, mouse PEPT1 mediates H+-coupled electrogenic transport of the dipeptide glycylsarcosine. The mouse pept1 gene, cloned from a genomic DNA library in bacterial artificial chromosome, is ∼38 kb long and consists of 23 exons and 22 introns. 5′-Rapid amplification of cDNA ends with poly(A)+ RNA from mouse intestine has identified the transcription start site that lies 31 bp upstream of the translation start site. The promoter region upstream of the transcription start site does not contain the TATA box but possesses three GC boxes which are the binding sites for the transcription activator SP1. Functional analysis of the promoter region using the luciferase reporter assay in Caco-2 cells (a human intestinal cell line that express PEPT1 constitutively) and five different 5′-deletion fragments of the promoter has shown that essential promoter/enhancer elements are present within 1140 bp upstream of the transcription start site.