It has been previously demonstrated that rhythmically firing neurons (Pre-I neurons) preceding cervical root (C4 or C5) inspiratory activity, localized in the rostral ventrolateral medulla (RVL), are important in the generation of the basic respiratory rhythm in brainstem-spinal cord preparations from newborn rats. We examined the effects of single and continuous electrical stimulation applied to the RVL on Pre-I and C4 activities in these preparations. We verified that the phase of respiratory rhythm was reset when Pre-I firing was induced in both right and left RVL by single shock stimulation, whether C4 activity appeared or not. Lower frequency and intensity of continuous pulse train stimulation in the RVL enhanced Pre-I activity, and hence C4 activity, whereas higher frequency and intensity inhibited both. The results suggest that synchronous burst activity between the right and left Pre-I neurons must be above a certain level (in its intraburst firing rate) to trigger C4 inspiratory activity and, therefore, that cooperation among Pre-I neurons is important for induction of rhythmic inspiratory drive. After bilateral lesions of the caudal ventrolateral medulla, Pre-I neurons retained their rhythmic activity, while C4 activity disappeared. Present results further confirmed our hypothesis that Pre-I neurons are the primary generator of respiratory rhythm. We propose a hypothetical model of the generation of rhythmic respiratory activity.