Since human papillomavirus (HPV) infection was first identified as a risk factor for cervical cancer, several seroepidemiologic and tissue-based studies have investigated HPV in relation to prostate cancer, another common genitourinary malignancy, with mixed results. To further inform this potential association, we conducted a large, prospective investigation of HPV types 16, 18, and 31 in relation to risk of prostate cancer in the Prostate Cancer Prevention Trial. Cases were a sample of men diagnosed with prostate cancer after visit 2 or on their end-of-study biopsy (n = 616). Controls were men not diagnosed with prostate cancer during the trial or on their end-of-study biopsy (n = 616). Controls were frequency matched to cases by age, treatment arm, and family history of prostate cancer. Sera from visit 2 were tested for IgG antibodies against HPV types 16, 18, and 31. No associations were observed for weak or strong HPV-16 [odds ratio (OR), 0.94; 95% confidence interval (95% CI), 0.53-1.64 and OR, 1.07; 95% CI, 077-1.48, respectively], HPV-18 (OR, 0.75; 95% CI, 0.27-2.04 and OR, 0.87; 95% CI, 0.47-1.63, respectively), or HPV-31 seropositivity (OR, 0.76; 95% CI, 0.45-1.28 and OR, 1.15; 95% CI, 0.80-1.64, respectively) and risk of prostate cancer. Considering this finding in the context of the HPV and prostate cancer literature, HPV does not appear to be associated with risk of prostate cancer, at least by mechanisms proposed to date, and using epidemiologic designs and laboratory techniques currently available.