The mesencephalon, especially the periaqueductal gray, is believed to integrate specific movement patterns of the somatic and autonomic nervous system, including those of vocalization, defensive behaviors and others. Fiber communications exist between the periaqueductal gray and the pontine micturition center, and many nerve fibers ascending from the sacral spinal cord project to the periaqueductal gray. We examined whether the mesencephalon is involved in micturition function using microstimulation and a neurotracer. We decerebrated 28 adult cats under general anesthesia. An electrode that can be used for microinjection was positioned in stereotaxic fashion in the mesencephalon and pons. Subsequently electrical stimulation and chemical stimulation with DL-homocysteine acid were applied to search for micturition inducing sites. Blood pressure and respiration were monitored simultaneously. We also performed electrical microstimulation of pontine micturition center. The neurotracer 5% Fluoro-Gold (Denver, Colorado) was injected into these sites to identify neural pathways between the mesencephalon and pons. The brainstem was removed after 10 hours and the mesencephalon was examined by fluorescence microscopy. Bladder contraction was provoked by electrical and chemical stimulation applied mainly at the ventrolateral side of the periaqueductal gray. Blood pressure increased simultaneously with bladder contraction after periaqueductal gray stimulation. Neurotracer injected into the pontine micturition center was found mainly on the ventrolateral side of the periaqueductal gray, in agreement with the sites where micturition was provoked by microstimulation. Nerve cells on the ventrolateral side of the periaqueductal gray have neural communications with the pontine micturition center bilaterally and they regulate micturition.