Women with high androgen levels appear to be at increased risk for breast cancer. The 5-alpha-reductase type 2 enzyme (SRD5A2) is an important mediator of local androgen actions. The SRD5A2 gene contains a polymorphism leading to a valine to leucine change in codon 89 (V89L). The Leu allele has been associated with lower SRD5A2 activity and might be protective for breast cancer. At the same time, among breast cancer patients, the Leu allele has been associated with lower prostate-specific antigen expression, indicating poor prognosis. Within a cohort of breast cancer screening participants in the Netherlands (DOM-cohort) we examined whether the V89L polymorphism is associated with the risk and prognosis of breast cancer. We studied 295 postmenopausal breast cancer cases and a randomly selected reference group from the baseline cohort (n = 382). The genotype distribution in the reference group was: VV 52%; VL 40%; and LL 8%. Compared with women with the VV genotype, adjusted breast cancer rate ratios for women with VL and LL genotypes were 1.5 (95% confidence interval = 1.0-2.2) and 1.1 (95% confidence interval = 0.5-2.1), respectively. Compared with breast cancer patients with VV or VL genotypes, those with the LL genotype showed larger tumors (proportion with size > 2 cm is 26 versus 55%, respectively; P = 0.07), a higher frequency of positive lymph nodes (28 versus 55%, respectively; P = 0.09), and a higher tumor-node-metastasis stage (proportion with stage III/IV: 6 versus 25%, respectively; P = 0.04). The LL genotype is also associated with shorter survival than the VV and VL genotypes (P = 0.10). In conclusion, our findings do not provide evidence for an important role of the V89L polymorphism in the etiology of breast cancer. However, in breast cancer patients, the LL genotype may be associated with unfavorable prognosis.