Objective: We evaluated the value of transperineal ultrasound-guided template saturation biopsies (TTSB) in patients in whom prostate cancer was suspected despite previously negative transrectal prostate biopsies. Materials and methods: The study included 15 patients (mean age 62 years; range 54 to 76 years) who underwent TTSB. The total number of repeat negative biopsies was 43, ranging from 1 to 5, and biopsy cores ranging from 6 to 27. Five patients received 5-alpha-reductase inhibitors before TTSB. All the patients had persistently high prostate specific antigen levels (mean 9.4 ng/ml; range 2.5 to 22.1 ng/ml). Digital rectal examination was positive in three patients. One patient had high-grade prostatic intraepithelial neoplasia in the last biopsy. The mean duration from the first biopsy to TTSB was 3.8 years (range 6 months to 9 years). Before TTSB, a transurethral catheter was inserted following antibiotic prophylaxis. Biopsies were performed in lithotomy position under general or spinal anesthesia. A biplanar transrectal ultrasound probe was attached to a brachytherapy unit and a brachytherapy template with holes 0.5 cm apart was positioned over the perineum. Biopsy cores were obtained by a transperineal approach through the template grids using an 18-gauge biopsy needle. The patients were discharged the day after the procedure. Results: The mean number of biopsy cores obtained by the transperineal saturation biopsy technique was 55.6 (range 36 to 87). Adenocarcinoma was detected in two patients (13.3%). Two patients experienced transient urinary retention after removal of the urethral catheter. No other complications occurred. Conclusion: The results of this preliminary study show that TTSB is a safe procedure enabling adequate sampling of the prostate in patients having risk for prostate cancer despite previous negative repeat biopsies.