Transrectal ultrasound-guided needle biopsy of the prostate is routinely performed to diagnose prostate cancer. We performed a prospective study to assess the pain and identify risk factors of pain during prostate biopsy. Prospectively, 131 patients were enrolled. Transrectal ultrasound-guided needle prostate biopsies were performed without any anesthesia. Pain was assessed by using an immediate postbiopsy anonymous questionnaire including a linear visual analog scale (VAS). Six factors were studied (age, prostate volume, cores number, operator, previous biopsy and first core location). Most of the patients tolerated the biopsy with acceptable discomfort. Among the risk factors studied, only first core location influenced the pain. Apex biopsy first was more painful. We recommend starting biopsy with the base.