Unilateral microinjection of carbachol (CCh, 1.0 microg/0.2 microl) into the ventrolateral periaqueductal gray matter (vPAG) increased the duration of tonic immobility (TI) episodes induced by postural inversion and by movement restriction maneuvers in adult male guinea pig (Cavia porcellus), while stimulation with the same drug at the same concentration into the dorsolateral and dorsomedial periaqueductal gray matter (dl/dmPAG) decreased the duration of TI. Pretreatment with atropine (7.6 microg/0.4 microl) showed that the action of CCh is mediated by muscarinic receptors in the ventrolateral PAG but not in the dorsomedial and dorsolateral regions. These data suggest that the PAG and the cholinergic system are involved in the modulation of TI episodes and that different regions of the guinea pig PAG play distinct roles in the organization of this behavior.