Endogenous carbon monoxide (CO) produced by heme oxygenase (HO) is reported as a relaxation factor in the urethral smooth muscle, while roles of CO in the brain in regulation of the micturition reflex remain unclear. In this study, to elucidate roles of brain endogenous CO in regulation of the micturition reflex, we investigated effects of centrally administered CORM3 (CO donor) and ZnPP (non-selective HO inhibitor) on the micturition reflex in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. A catheter was inserted into the bladder to perform cystometrograms (CMG), which was started 2 h after the surgery, and 1 h after the start, CORM3 or ZnPP was intracerebroventricularly (icv) administered. CORM3 (1 or 10 nmol/rat, icv) dose-dependently prolonged intercontraction intervals (ICI), while ZnPP (10 or 30 nmol/rat, icv) dose-dependently shortened ICI. The ZnPP (30 nmol/rat, icv)-induced ICI shortening was reversed in the presence of CORM3 (10 nmol/rat, icv). In addition, ZnPP (30 nmol/rat, icv) significantly reduced single-voided volume and bladder capacity without affecting post-voiding residual volume or voiding efficiency. These results suggest that brain endogenous CO can inhibit the micturition reflex in rats.