Breathing is highly sensitive to the PCO of arterial blood. Although CO is detected via the proxy of pH, CO acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO-binding to Cx26 to devise a dominant negative subunit (Cx26) that removes the CO-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26 in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area - reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO. As central chemosensors mediate about 70% of the total response to hypercapnia, CO-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO. Our data unequivocally link the direct sensing of CO to the chemosensory control of breathing and demonstrates that CO-binding to Cx26 is a key transduction step in this fundamental process.