Focal hypoxia in the pre-Botzinger complex (pre-BotC) in vivo elicits excitation of inspiratory motor output by modifying the patterning and timing of phrenic bursts. Hypoxia, however, has been reported to enhance glutamate release in some regions of the brain, including the medullary ventral respiratory column; thus, the pre-BotC-mediated hypoxic respiratory excitation may result from, or be influenced by, hypoxia-induced activation of ionotropic glutamate (i.e., excitatory amino acid, EAA) receptors. To test this possibility, the effects of focal pre-BotC hypoxia (induced by sodium cyanide, NaCN) were examined before and after blockade of ionotropic EAA receptors (using kynurenic acid, KYN) in this region in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Before blockade of ionotropic EAA receptors, unilateral microinjection of NaCN (1 mM; 10-20 nl) into the pre-BotC produced either phasic or tonic excitation of phrenic nerve discharge (as previously described). Unilateral microinjection of KYN (50-100 mM; 40 nl) decreased the amplitude and frequency of basal phrenic nerve discharge, however, subsequent microinjection of NaCN, but not DL-homocysteic acid (DLH, a glutamate analog), still produced excitation of phrenic motor output. Under these conditions, the NaCN-induced excitation included frequency modulation of phasic phrenic bursts, and in many cases, augmented and/or fractionated phrenic bursts. These findings demonstrate that the hypoxia sensing function of the in vivo pre-BotC, which produces excitation of phrenic nerve discharge, is not dependent upon activation of ionotropic glutamate receptors, but ionotropic glutamate receptor activation may modify the expression of the focal hypoxia-induced response. Thus, the current findings provide additional support to the concept of intrinsic hypoxic sensitivity of the pre-BotC.