Direct projections from the periaqueductal gray (PAG) to the pontine micturition center neurons directly projecting to the lumbosacral cord segments were observed electron microscopically in the rat by a double labeling method. Biotinylated dextran amine (BDA) was injected into the PAG and horseradish peroxidase (HRP) was injected into the lumbosacral cord segments. After injection of BDA into the ventrolateral part of the PAG, many BDA-labeled axons were seen light microscopically in Barrington's nucleus; a moderate number of them were found in the pontine tegmental region just ventral to Barrington's nucleus (D-region [Ding, Y-Q., Takada, M., Tokuno, H. and Mizuno, N., J. Comp. Neurol., 357 (1996) 318-330]). On the other hand, after injection of BDA into the dorsolateral part of the PAG, only a few BDA-labeled axons were seen in Barrington's nucleus or the D-region. BDA-labeled axon terminals were electron microscopically confirmed to be in synaptic contact with HRP-labeled dendrites and somata in Barrington's nucleus and the D-region. The results indicate that the ventrolateral part of the PAG is implicated in regulation of the micturition reflex.