Sex steroid hormones influence prostate development and maintenance through their roles in prostate cellular proliferation, differentiation and apoptosis. Although suspected to be involved in prostate carcinogenesis, an association between circulating androgens and prostate cancer has not been clearly established in epidemiologic studies. We conducted a nested case-control study with prospectively collected samples in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, to examine associations of prostate cancer with androstenedione (Δ4-A), testosterone (T), sex hormone-binding globulin (SHBG) and 3α-androstanediol glucuronide (3α-diolG). A total of 727 incident Caucasian prostate cancer cases (age ≥ 65 years, N = 396) and 889 matched controls were selected for this analysis. Overall, prostate cancer risks were unrelated to serum T, estimated free and bioavailable T, and SHBG; however, risks increased with increasing T:SHBG ratio (ptrend = 0.01), mostly related to risk in older men (≥65 years, ptrend = 0.001), particularly for aggressive disease [highest versus lowest quartile: odds ratio (OR) 2.76, 95% confidence interval (CI) 1.50–5.09]. No clear patterns were noted for Δ4-A and 3α-diolG. In summary, our large prospective study did not show convincing evidence of a relationship between serum sex hormones and prostate cancer. T:SHBG ratio was related to risk in this older population of men, but the significance of this ratio in steroidal biology is unclear. © 2008 Wiley-Liss, Inc.