The purpose of the present study was to determine whether murines could be substituted for traditional experimental mammals to study the brainstem mechanism of vocalization. We conducted systematic electrical and chemical stimulation of the brainstem in guinea pigs to identify the similarities in the call sites between murines and other mammals. We further examined whether or not fictive vocalization could be induced in paralyzed guinea pigs, an experimental model which facilitates neuronal recording in the brainstem. The sites where electrical stimulation evoked vocalization were distributed continuously from the periaqueductal grey (PAG) to the lower brainstem. This call area usually ended at the most caudal part of the inferior olive and thus did not continuously extend to the nucleus retroambiguus. Microinjections of d,l-homocysteic acid and bicuculline induced vocalization at the PAG, parabrachial nucleus, and the most dorsal part of the pontine reticular formation. The brainstem call areas and vocal motor patterns induced from these areas were approximately consistent with those in other mammals. Fictive vocalization induced by PAG stimulation could be identified from activities of the phrenic, abdominal, and superior laryngeal nerves in paralyzed guinea pigs. We thus concluded that guinea pigs can be utilized in studies of brainstem vocal mechanism.