The nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain located between the roof of the midbrain and the cerebellum. From a neuroanatomical perspective, the NI can be compared with the pons which, in mammals, contributes to the control of breathing pattern. This study tested the hypothesis that the NI plays a critical role in breathing pattern formation in the bullfrog. More specifically, we postulated that this nucleus was the site responsible for clustering breaths into distinct episodes of breathing. This hypothesis was tested by comparing the respiratory motor output of decerebrate, paralyzed and artificially ventilated bullfrogs before and after bilateral lesions of the NI by pressure microinjections of lidocaine or kainic acid (KA) into this area. Bilateral microinjections of lidocaine or KA into the NI transformed the breathing pattern from episodic (many breaths per episode) to one of evenly spaced single breaths, without affecting the amplitude of the fictive breaths. These changes in breathing pattern were associated with an overall decrease in breathing frequency and a reduction in CO2-chemosensitivity. Breathing episodes of more than one breath reappeared during hypercarbia (3.5% CO2 in air) after KA lesioning. Bilateral lesions to the NI did not affect the changes in the timing or the amplitude of the respiratory-related bursts elicited by pulmonary stretch receptor feedback, indicating that mechanoreflexes do not require NI input. We conclude that the NI is not responsible for the genesis of breathing episodes, but provides a tonic excitatory input to respiratory centers in the lower brainstem. The NI also plays an important role in either CO2 chemodetection or, more probably, integration of CO2 chemoreceptor information. This, in turn, contributes to the production of episodes of more than one breath.