Parkinson's disease is a progressive neurodegenerative disorder characterized clinically by rigidity, akinesia, resting tremor and postural instability. It has recently been suggested that low frequency stimulation of the pedunculopontine nucleus (PPN) has a role in the therapy for Parkinsonism, particularly in gait disorder and postural instability. However, there is limited information about the mechanism of low frequency stimulation of the PPN on Parkinson's disease. The present study was to investigate the effect and mechanism of low frequency stimulation of the PPN on the firing rate of the ventrolateral thalamic nucleus (VL) in a rat model with unilateral 6-hydroxydopamine lesioning of the substantia nigra pars compacta. In vivo extracellular recording and microiontophoresis were adopted. The results showed that the firing rate of 60.71% VL neurons in normal rats and 59.57% VL neurons in 6-hydroxydopamine lesioned rats increased with low frequency stimulation of the PPN. Using microiontophoresis to VL neurons, we found the firing rate in VL neurons responded with either an increase or decrease in application of acetylcholine (ACh) in normal rats, whereas with a predominant decrease in M receptor antagonist atropine. Furthermore, the VL neurons were mainly inhibited by application of γ-aminobutyric acid (GABA) and excited by GABA(A) receptor antagonist bicuculline. Importantly, the VL neurons responding to ACh were also inhibited by application of GABA. We also found that the excitatory response of the VL neurons to the low frequency stimulation of the PPN was significantly reversed by microiontophoresis of atropine. These results demonstrate that cholinergic and GABAergic afferent nerve fibers may converge on the same VL neurons and they are involved in the effects of low frequency stimulation of the PPN, with ACh combining M(2) receptors on the presynaptic membrane of GABAergic afferents, which will inhibit the release of GABA in the VL and then improve the symptoms of Parkinson's disease.