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Animal and laboratory studies suggest that regular use of non-steroidal anti-inflammatory drugs (NSAIDs) may reduce prostate cancer risk. The aim of this study was to investigate the association between NSAID use and prostate cancer in a high-risk population. We included 1299 men who were referred to our university's prostate cancer detection clinic for prostate biopsy between January 1999 and July 2003. Before transrectal ultrasonography and prostate biopsy, all men completed a self-administered questionnaire that included questions on drug use in the preceding 5 years. On average, NSAID users were older than non-users but there was no significant difference in mean baseline prostate-specific antigen (PSA). Four hundred and ninety-four (38%) had biopsy-confirmed prostate cancer. After adjustment for age, family history of prostate cancer and other potential confounders, use of aspirin was associated with a 42% reduction in the odds of prostate cancer detection [95% confidence interval (CI) 0.36–0.91]. Among cases, regular use of NSAIDs was inversely related to the risk of detection of more poorly differentiated cancers and cancers with higher percentage core involvement. These findings support other epidemiological and experimental evidence that suggests that aspirin may be useful in prostate cancer prevention. Further observational studies with adequate case definition and exposure measurements and careful adjustment for detection bias are warranted.