We demonstrate the feasibility of a novel and nonionizing process for bladder imaging in vivo, called photoacoustic cystography (PAC). Using a photoacoustic imaging system, we have successfully imaged a rat bladder filled with clinically used Methylene Blue (MB) dye. An image contrast of ∼8 was achieved. Further, spectroscopic PAC confirmed the accumulation of MB in the bladder. Using a laser pulse energy of less than 1 mJ=cm 2 (1=20 of the ANSI safety limit), a deeply (1:2 cm) positioned bladder in biological tissues was clearly visible in the PA image. Our results suggest that PAC can potentially provide a nonionizing, relatively cheap, and portable tool for bladder mapping. Among our clinical interests, nonionizing PAC with an injection of MB can potentially monitor vesicoureteral reflux in children.