Sex hormones have been implicated in prostate carcinogenesis and are thought to modulate cell proliferation and growth. To investigate the association between polymorphisms in hormone-related genes and prostate cancer risk, we conducted a two-stage, case-control study within the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Using DNA extracted from blood specimens, we initially genotyped 14 single nucleotide polymorphisms in genes involved in hormone regulation or metabolism (AKR1C3, CYP1A1, CYP1B1, CYP3A4, ESR1, GNRH1, HSD173B, HSD3B2, SHBG, and SRD5A2) in 488 prostate cancer cases and 617 matched controls. Heterozygotes at SHBG D356N were found to be associated with an increased risk of prostate cancer compared with the homozygous wild type, particularly among non-Hispanic whites (odds ratio, 1.54; 95% confidence interval, 1.13-2.09; P = 0.006). No significant associations were observed with the other polymorphisms. The SHBG D356N polymorphism, which has potential functional significance, was subsequently genotyped in additional 769 cases and 1,168 controls. Overall, SHBG D356N heterozygotes were found to have an increased risk of prostate cancer among whites (odds ratio, 1.34; 95% confidence interval, 1.10-1.63; P = 0.0007). This study suggests that genetic variation in SHBG may influence prostate cancer susceptibility.