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