We developed a local anesthetic procedure for three-dimensional 26-core prostate biopsy (3D26PBx), a combination of transperineal 14-core biopsy (TP14PBx) and transrectal 12-core biopsy (TR12PBx). At first, a periapical triangle, confined by the levator ani, the rhabdosphincter and the external anal sphincter muscle, was made visible by transrectal ultrasound. After administration of 1 mL of 1%-lidocaine into the midline perineal skin 1.5 cm above the anus, we inserted a spinal needle toward the periapical triangle for injection of 1.5-2.0 mL of 1%-lidocaine and performed the TP14PBx. After administration of the periprostatic nerve block with 10 mL of 1%-lidocaine, we performed the TR12PBx. The efficacy of the procedure was evaluated prospectively in 45 consecutive men undergoing the 3D26PBx. The 3D26PBx was completed with just local anesthesia in all patients. The pain levels, assessed by an 11-point visual analog scale, were not different between the TP14PBx and the TR12PBx.