This study examined cerebrovascular reactivity and ventilation during step changes in CO2 in humans. We hypothesized that: (1) end-tidal PCO2 (PET,CO2) would overestimate arterial PCO2 (Pa,CO2) during step variations in PET,CO2 and thus underestimate cerebrovascular CO2 reactivity; and (2) since PCO2 from the internal jugular vein (Pjv,CO2) better represents brain tissue PCO2, cerebrovascular CO2 reactivity would be higher when expressed against Pjv,CO2 than with Pa,CO2, and would be related to the degree of ventilatory change during hypercapnia. Incremental hypercapnia was achieved through 4 min administrations of 4% and 8% CO2. Incremental hypocapnia involved two 4 min steps of hyperventilation to change PET,CO2, in an equal and opposite direction, to that incurred during hypercapnia. Arterial and internal jugular venous blood was sampled simultaneously at baseline and during each CO2 step. Cerebrovascular reactivity to CO2 was expressed as the percentage change in blood flow velocity in the middle cerebral artery (MCAv) per mmHg change in Pa,CO2 and Pjv,CO2. During hypercapnia, but not hypocapnia, PET,CO2 overestimated Pa,CO2 by +2.4 ± 3.4 mmHg and underestimated MCAv-CO2 reactivity (P < 0.05). The hypercapnic and hypocapnic MCAv-CO2 reactivity was higher (∼97% and ∼24%, respectively) when expressed with Pjv,CO2 than Pa,CO2 (P < 0.05). The hypercapnic MCAv-Pjv,CO2 reactivity was inversely related to the increase in ventilatory change (R2 = 0.43; P < 0.05), indicating that a reduced reactivity results in less central CO2 washout and greater ventilatory stimulus. Differences in the PET,CO2, Pa,CO2 and Pjv,CO2-MCAv relationships have implications for the true representation and physiological interpretation of cerebrovascular CO2 reactivity.