The authors conducted a nested case-control study of serum steroid concentrations and risk of benign prostatic hyperplasia (BPH), using data from the placebo arm of the Prostate Cancer Prevention Trial (1993–2003). Incident BPH over 7 years (n=708) was defined as receipt of treatment, a report of 2 International Prostate Symptom Score (IPSS) values greater than 14, or 2 increases of 5 or more from baseline IPSS values with at least 1 value greater than or equal to 12. Controls (n=709) were selected from men who reported no BPH treatment or any IPSS greater than 7. Baseline serum was analyzed for testosterone, estradiol, estrone, 5α-androstane-3α, 17β-diol-glucuronide, and sex hormone-binding globulin. Covariate-adjusted odds ratios contrasting the highest quartiles with the lowest quartiles of testosterone, estradiol, and testosterone:17β-diol-glucuronide ratio were 0.64 (95% confidence interval (CI): 0.43, 0.95; Ptrend=0.04), 0.72 (95% CI: 0.53, 0.98; Ptrend=0.09), and 0.64 (95% CI: 0.46, 0.89; Ptrend=0.004), respectively. Findings did not differ by age, body mass index, time to BPH endpoint, or type of BPH endpoint. High testosterone levels, estradiol levels, and testosterone:17β-diol-glucuronide ratio are associated with reduced BPH risk, which may reflect decreased activity of 5-α-reductase. Genetic or environmental factors that affect the activity of 5-α-reductase may be important in the development of symptomatic BPH.