The human equilibrative nucleoside transporter 1 gene (hENT1) is the primary nucleoside transporter for cytosine arabinoside (AraC), a deoxycytidine analog used for treatment of acute leukemias and lymphomas. We screened approximately 1.6 kb upstream of the transcription initiation site of hENT1 for single nucleotide polymorphisms (SNPs) that affect gene expression. We identified one SNP at position -706G>C with a frequency of 21% in whites and 5% in African-Americans. In African-Americans, we observed two SNPs at positions -1345C>G and -1050G>A with allele frequencies of 8% and 19%, respectively. TRANSFAC analysis suggested that -1345C>G and -706G>C may alter transcription factor binding sites. Four naturally occurring haplotypes (CGG, CAG, CGC and GAG) were cloned into a luciferase expression plasmid, transfected into Cos-1 cells, and reporter activity measured at 24 and 48 h. Three haplotypes, CAG, CGC and GAG, respectively, showed average expression that was approximately two-fold (P<0.05), 1.4-fold (P<0.05) and 1.1-fold (P>0.05) higher than lowest expression haplotype CGG at 48 h. When reanalysed as single SNPs, the differences in expression were significant for -1345C>G and -1050G>A genotypes, and not for -706G>C. However, the magnitude of difference was reduced, suggesting that no single SNP completely accounts for the expression differences observed at the haplotype level. By real-time quantitative reverse transcriptase-polymerase chain reaction assay, individuals with CGG/CGC haplotypes showed 1.37-fold higher median expression of hENT1 transcript than those with common CGG/CGG haplotypes. Although not statistically significant (P=0.12), this difference is in the direction predicted by the in vitro data. hENT1 promoter region haplotypes may influence gene expression and alter AraC chemosensitivity.