We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C, About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5'-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5'-flanking region showed that the nucleotide sequence from -652 to -111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (-296/-303, -307/ -313) and two MAZ binding sites (-353/-359, -522/ -528) in this region. Luciferase assay results suggest that MAZ and Sp-l transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.