Folate status may affect cancer risk through its role in both methylation and nucleotide synthesis of DNA. A low dietary intake of folate has been linked to risk of several cancers, but epidemiologic studies with reference to prostate cancer are scanty. We therefore analyzed data from a case-control study of prostate cancer conducted between 1991 and 2002 in various areas of Italy. Cases were 1,294 patients with incident, histologically confirmed prostate cancer and controls were 1,451 patients admitted to the same network of hospitals of cases for acute, nonneoplastic conditions. All subjects were <75 years old. Intake of folate and other nutrients was computed from a validated food frequency questionnaire. We adjusted for energy intake using the residual method, and calculated multivariate odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression. The OR of prostate cancer was 0.66 (95% CI, 0.51-0.85) for the highest versus the lowest quintile of folate intake. The relation between dietary folate and prostate cancer was consistent across strata of age, methionine, vitamin B6, and alcohol intake, and did not vary substantially according to Gleason score of prostate cancer. The combined OR for high-folate and low-alcohol intake versus low-folate and high-alcohol intake was 0.46 (95% CI, 0.29-0.75). Therefore, this study supports a favorable role of dietary folate on prostate cancer risk.