Neurokinin-1 receptor (NK1R)-expressing neurons that are involved in chemoreception at the retrotrapezoid nucleus (Nattie & Li, 2002b) are also prominent at locations that contain medullary serotonergic neurons, which are chemosensitive in vitro. In medullary regions containing both types, we evaluate their role in central chemoreception by specific cell killing. We inject (2 x 100 nl) a) substance P-saporin (SP-SAP; 1 uM) to kill NK1R-expressing neurons, b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti-SERT-SAP; 1 uM) to kill serotonergic neurons, or c) SP-SAP and anti-SERT-SAP together to kill both types. Controls receive IgG-SAP injections (1 uM). There is no double-labeling of NK1R-immunoreactive (ir) and tryptophan-hydroxylase (TPOH)-ir neurons. Cell (somatic profile) counts show that NK1R-ir neurons in the SP-SAP group are reduced by 31%; TPOH-ir neurons in the anti-SERT-SAP group by 28%; and NK1R-ir and TPOH-ir neurons, respectively, in the combined lesion group by 55% and 31% (P<0.001; two-way ANOVA; P<0.05, post-hoc Tukey test). The treatments had no significant effect on sleep/wake time, body temperature, or oxygen consumption but all three reduced the ventilatory response to 7% inspired CO2 in wakefulness and sleep by a similar amount. SP-SAP treatment decreased the averaged CO2 responses (3, 7, and 14 days after lesions) in wakefulness and sleep by 21% and 16%, anti-SERT-SAP by 15% and 18%, and the combined treatment by 12% and 12% (P<0.001; two-way ANOVA; P<0.05, post-hoc Tukey test). We conclude that separate populations of serotonergic and adjacent NK1R-expressing neurons in the medulla are both involved in central chemoreception in vivo.