The lateral periaqueductal gray (lPAG) is implicated in the expression of defensive behavior accompanied by various autonomic changes. On the other hand, the pedunculopontine tegmental nucleus (PPT), which is located in the caudal mesencephalic tegmentum, contains a large number of cholinergic neurons and is involved in various functions including the autonomic control. Previous retrograde tracing studies suggest that lPAG is one of the major sources of input to the PPT. It remains, however, unanswered whether or not cholinergic neurons in the PPT receive direct input from lPAG neurons. To address this issue, we examined the direct projections from the lPAG to PPT cholinergic neurons by using a combined anterograde tracing with biotinylated dextranamine (BDA) and immunohistochemistry for choline acetyltransferase (ChAT) that is a marker for cholinergic neurons. As a result, ChAT-immunoreactive (-ir) neurons in the PPT, especially in its pars compacta, were embedded in the plexus of fibers labeled with BDA injected into the ipsilateral lPAG. When the PPT pars compacta region was examined under an electron microscopy, the BDA-labeled axon terminals made asymmetrical synaptic contacts with dendrites and somata of the ChAT-ir neurons. Furthermore, in situ hybridization revealed that many of the lPAG neurons were positive for vesicular glutamate transporter 2 mRNA that is a marker for glutamatergic neurons, but not for glutamic acid decarboxylase 67 mRNA that is a marker for GABAergic neurons. These data suggest that glutamatergic lPAG neurons exert excitatory influence on the cholinergic neurons in the PPT pars compacta for the control of autonomic functions.