The pedunculopontine tegmental nucleus (PPTg) has been shown to have important functions relevant to the regulation of behavioral states and various motor control systems, including breathing control. The PPTg is considered an important nucleus in the mesopontine region with considerably cholinergic input to the ventral respiratory column. In addition, recent studies indicate that cholinergic innervation of the ventral respiratory column may play an important role in modulation of breathing. Here, we investigated the cholinergic stimulation of the PPTg and the changes in breathing output in conscious rats. Male Wistar rats (280-350 g, N = 5-12/group) with unilateral stainless steel cannula implanted into the PPTg were used. Respiratory parameters (tidal volume (VT), respiratory frequency (fR) and ventilation (VE)) were analyzed by whole body plethysmography. In unrestrained awake rats, unilateral injection of the cholinergic muscarinic agonist carbachol (10 mM-100 nL) in the PPTg decreased fR, and increase VT, without changing VE. The changes in fR and VT elicited by carbachol into the PPTg are abolished by previous blockade of the M4 muscarinic cholinergic receptors tropicamide into the PPTg. No significant changes in fR and VT elicited by carbachol were observed after blockade of the M1 and/or M3 muscarinic cholinergic receptors pirenzepine or 4-DAMP into the PPTg. Our data suggest that the changes in fR and VT produced by muscarinic cholinergic stimulation of PPTg is presumably mediated through a Gi-coupled M4 muscarinic receptors.