We evaluated the hypothesis that N-methyl-D-aspartate (NMDA) receptors in the rostral pons mediate the off-switch of inspiration in the adult rat. Experiments were performed on decerebrate, vagotomized, paralyzed and ventilated animals. Activity of phrenic nerve was recorded. Small volumes (10 nl) of NMDA antagonists, MK-801 and AP-5, or non-NMDA antagonists, CNQX and DNQX, were injected into the rostral pons. We found that injections of MK-801 reversibly increased the duration of neural inspiration (TI), and the increase was dose-dependent. Injections of AP-5 also increased TI. Injections of the DNQX and CNQX in these same loci resulted in no significant changes in the duration of neural inspiration, expiration or peak phrenic activity (PNA). However, injections of kainic acid (KA, 4.7 mM) in the loci increased TI and decreased PNA. We conclude that neurons regulating the off-switch mechanism are located in the rostral pons. Further, the binding of NMDA receptors in the rostral pons is involved in this off-switch mechanism.