Indomethacin (INDO), a reversible cyclooxygenase inhibitor, is a useful tool for assessing the role of cerebrovascular reactivity on ventilatory control. Despite this, the effect of INDO on breathing stability during wakefulness has yet to be examined. Although the effect of reductions in cerebrovascular CO(2) reactivity on ventilatory CO(2) sensitivity is likely dependent upon the method used, no studies have compared the effect of INDO on steady-state and modified rebreathing estimates of ventilatory CO(2) sensitivity. The latter method includes the influence of PCO(2) gradients and cerebral perfusion, whereas the former does not. We examined the hypothesis that INDO-induced reduction in cerebrovascular CO(2) reactivity would 1) cause unstable breathing in conscious humans and 2) increase ventilatory CO(2) sensitivity during the steady-state method but not during rebreathing methods. We measured arterial blood gases, ventilation (VE), and middle cerebral artery velocity (MCAv) before and 90 min following INDO ingestion (100 mg) or placebo in 12 healthy participants. There were no changes in resting arterial blood gases or Ve following either intervention. INDO increased the magnitude of Ve variability (index of breathing stability) during spontaneous air breathing (+4.3 +/- 5.2 Deltal/min, P = 0.01) and reduced MCAv (-25 +/- 19%, P < 0.01) and MCAv-CO(2) reactivity during steady-state (-47 +/- 27%, P < 0.01) and rebreathing (-32 +/- 25%, P < 0.01). The Ve-CO(2) sensitivity during the steady-state method was increased with INDO (+0.5 +/- 0.5 l x min(-1) x mmHg(-1), P < 0.01), while no changes were observed during rebreathing (P > 0.05). These data indicate that the net effect of INDO on ventilatory control is an enhanced ventilatory loop gain resulting in increased breathing instability. Our findings also highlight important methodological and physiological considerations when assessing the effect of INDO on ventilatory CO(2) sensitivity, whereby the effect of INDO-induced reduction of cerebrovascular CO(2) reactivity on ventilatory CO(2) sensitivity is unmasked with the rebreathing method.