Abstract Variation in the cytochrome P450 oxidoreductase (POR) gene, a key regulator of type II cytochrome P450 enzymes, may affect exposure to endogenous steroid hormones and breast cancer risk. We sequenced the POR locus and tested candidate polymorphisms G5G and A503V for association with breast cancer risk among women in the Multiethnic Cohort Study (1,615 cases and 1,962 controls). The single nucleotide polymorphism (SNP) A503V was common in all racial/ethnic populations (minor allele frequency, ≥0.05) but was not associated with risk. SNP G5G (A → G nucleotide change), which lies in a suggestive exonic splicing enhancer motif in exon 1, was common only in African Americans (minor allele frequency, 0.21) and the homozygous state was modestly associated with increased breast risk among all cases [345 cases and 426 controls; odds ratio (OR), 1.64; 95% confidence interval (CI), 0.89–3.04; P = 0.12] and among cases with advanced disease (95 cases: OR, 3.08; 95% CI, 1.42–6.70; P = 0.005). In an attempt to replicate this association, we genotyped SNP G5G in additional African American case-control studies (747 cases and 468 controls). Nonsignificant positive associations were noted with the GG genotype class in all studies. In the pooled analysis (1,038 cases and 877 controls with genotype data), the association was statistically significant among all cases (OR, 1.58; 95% CI, 1.04–2.41; P = 0.03) and stronger in those with advanced disease (411 cases and 877 controls; OR, 2.60; 95% CI, 1.56–4.34; P = 0.0002). These data suggest that African Americans harbor an allele at the POR locus that may increase breast cancer risk. [Cancer Res 2007;67(8):3565–8]