We investigated, in a midbrain parasagittal slice preparation of Wistar rats (postnatal day 9–17), the synaptic inhibition of neurons in the pedunculopontine tegmental nucleus (PPN), which was mediated by gamma (γ)-amino-butyric acid (GABA). Whole-cell patch-clamp recording was used, in combination with a single-cell reverse transcription-polymerase chain reaction amplification technique, to record synaptic potentials and to identify the phenotype of the recorded PPN neuron. In the presence of the ionotropic glutamate receptor antagonists, 6-cyano-2, 3-dihydroxy-7-nitro-quinoxaline-2, 3, dione, and dl-2-amino-5-phosphonovaleric acid, single electrical stimuli were applied to the substantia nigra pars reticulata (SNr), one of the basal ganglia output nuclei. Stimulation of the SNr evoked inhibitory postsynaptic potentials (IPSPs) in 73 of the 104 neurons in the PPN. The IPSPs were abolished with a GABAA receptor antagonist, bicuculline. Inhibitory postsynaptic currents of the neurons were reversed in polarity at approximately −93.5 mV, which was close to the value of the equilibrium potential for chloride ions of −88.4 mV. Single-cell reverse transcription-polymerase chain reactions revealed that approximately 30% (9/32) of the PPN neurons that received inhibition from the SNr expressed detectable levels of choline acetyltransferase mRNA. These findings show that output from the SNr regulates the activity of cholinergic PPN neurons through GABAA receptors.