Neuronal recordings, microstimulation, and electrolytic and chemical lesions were used to examine the involvement of the Bötzinger Complex (BötC) in the bilateral phrenic-to-phrenic inhibitory reflex. Experiments were conducted in decerebrate cats that were paralyzed, ventilated, thoracotomized, and vagotomized. Microelectrode recordings within the BötC region revealed that some neurons were activated by phrenic nerve stimulation (15 of 69 expiratory units, 9 of 67 inspiratory units, and 19 nonrespiratory-modulated units) at average latencies similar to the onset latency of the phrenic-to-phrenic inhibition. In addition, microstimulation within the BötC caused a short latency transient inhibition of phrenic motor activity. In 17 cats phrenic neurogram responses to threshold and supramaximal (15 mA) stimulation of phrenic nerve afferents were recorded before and after electrolytic BötC lesions. In 15 animals the inhibitory reflex was attenuated by bilateral lesions. Because lesion of either BötC neurons or axons of passage could account for this attenuation, in eight experiments the phrenic-to-phrenic inhibitory responses were recorded before and after bilateral injections of 5 microM kainic acid (30-150 nl) into the BötC. After chemical lesions, the inhibitory response to phrenic nerve stimulation remained; however, neuronal activity typical of the BötC could not be located. These results suggest that axons important in producing the phrenic-to-phrenic reflex pass through the region of the BötC, but that BötC neurons themselves are not necessary for this reflex.