To test the physiological significance of CO2-sensitive neurons identified in the NTS, we measured the HCVR using barometric pressure plethysmography on unrestrained rats breathing 0, 5 and 7% CO2 in 21% O2 after bilateral NTS lesions with microinjection (200 nL) of substance P-saporin conjugates (SP-SAP, n = 15). For controls we microinjected a conjugate of saporin and an 11 amino acid nonsense peptide (blank, n = 9). Because chronic hypoxia also affects CO2-sensitivy, we studied rats acclimatized to PIO2 = 70 Torr for 7 days (CH; SP-SAP n = 10; blank n = 7). ANOVA showed significant effects of SP-SAP, CH and inspired CO2 on ventilation, with a significant interaction between SP-SAP and inspired CO2. SP-SAP decreased the HCVR mainly by an effect on tidal volume, as previously reported for SP-saporin lesions in the retrotrapezoid nucleus (J. Physiol. 544:603–616, 2002). Immunohistochemistry for the Neurokinin-1 receptor is being used to identify the nature and extent of SP-SAP lesions. Results show a physiological role for chemoreceptors in the NTS but more experiments are necessary to decide if chemoreceptors in the NTS versus other sites play a unique role in ventilatory acclimatization to hypoxia.