In this study we investigated possible separate effects of H⁺ ions and CO₂ on hypoxic sensitivity in humans. We also examined whether hypoxic sensitivity, conventionally defined as the ratio of (hypoxic -normoxic) ventilation over (hypoxic-normoxic) Hb oxygen saturation can also be estimated by taking the ratio (hypoxic-normoxic) ventilation over (log PaO₂ hypoxia - logPaO2normoxia), enabling one to measure the hypoxic response independently from potential confounding influences of changes in position of the Hb oxygen saturation curve. We used acetazolamide to induce a metabolic acidosis. To determine the acute hypoxic response (AHR) we performed step decreases in end-tidal PO₂ to ~50 Torr lasting 5 min each at three different constant end-tidal PCO₂ levels. Nine subjects ingested 250 mg acetazolamide or placebo every 8 hours for three days in a randomized double blind cross-over design. The metabolic acidosis was accompanied by a rise in ventilation, a substantial fall in PaCO₂ and a parallel leftwards shift of the ventilatory CO₂ response curve. In placebo, CO₂ induced equal relative increases in hypoxic sensitivity (O₂-CO₂ interaction) regardless of the way it was defined. Acetazolamide shifted the response line representing the relationship between hypoxic sensitivity and arterial [H⁺] ([H⁺_a]) to higher values of [H⁺_a] without altering its slope indicating that it did not affect the O₂-CO₂ interaction. So in contrast to earlier belief CO₂ and H⁺ have separate effects on hypoxic sensitivity. This was also supported by the finding that infusion of bicarbonate caused a leftwards shift of the hypoxic sensitivity-[H⁺_a] response lines in placebo and acetazolamide. A specific inhibitory effect of acetazolamide on hypoxic sensitivity was not demonstrated.