Reductions in end-tidal Pco₂ (Pet_CO2) during upright posture have been suggested to be the result of hyperventilation and the cause of decreases in cerebral blood flow (CBF). The goal of this study was to determine whether decreases in Pet_CO2 reflected decreases in arterial Pco₂ (Pa_CO2) and their relation to increases in alveolar ventilation (V̇a) and decreases in CBF. Fifteen healthy subjects (10 women and 5 men) were subjected to a 10-min head-up tilt (HUT) protocol. Pa_CO2, V̇a, and cerebral flow velocity (CFV) in the middle and anterior cerebral arteries were examined. In 12 subjects who completed the protocol, reductions in Pet_CO2 and Pa_CO2 (−1.7 ± 0.5 and −1.1 ± 0.4 mmHg, P < 0.05) during minute 1 of HUT were associated with a significant increase in V̇a (+0.7 ± 0.3 l/min, P < 0.05). However, further decreases in Pa_CO2 (−0.5 ± 0.5 mmHg, P < 0.05), from minute 1 to the last minute of HUT, occurred even though V̇a did not change significantly (−0.2 ± 0.3 l/min, P = not significant). Similarly, CFV in the middle and anterior cerebral arteries decreased (−7 ± 2 and −8 ± 2%, P < 0.05) from minute 1 to the last minute of HUT, despite minimal changes in Pa_CO2. These data suggest that decreases in Pet_CO2 and Pa_CO2 during upright posture are not solely due to increased V̇a but could be due to ventilation-perfusion mismatch or a redistribution of CO₂ stores. Furthermore, the reduction in Pa_CO2 did not fully explain the decrease in CFV throughout HUT. These data suggest that factors in addition to a reduction in Pa_CO2 play a role in the CBF response to orthostatic stress.