Catecholamine (CAT) release from adrenomedullary chromaffin cells (AMC) in response to stressors such as low O2 (hypoxia) and elevated CO2/H+ is critical during adaptation of the newborn to extrauterine life. Using a surrogate model based on a v -myc immortalized adrenal chromaffin cell line (i.e., MAH cells), combined with genetic perturbation of mitochondrial function, we tested the hypothesis that functional mitochondria are required for O2 sensing. Wild-type MAH cells responded to both hypoxia and increased CO2 (hypercapnia) with K+ current inhibition and membrane depolarization. Additionally, these stimuli caused a rise in cytosolic Ca2+ and CAT secretion, determined by fura-2 spectrofluorimetry and carbon fiber amperometry, respectively. In contrast, mitochondria-deficient (ρ0) MAH cells were hypoxia insensitive, although responses to hypercapnia and expression of several markers, including carbonic anhydrase II, remained intact. Rotenone (1 μM), a mitochondrial complex I blocker known to mimic and occlude the effects of hypoxia in primary AMC, was effective in wild-type but not ρ0 MAH cells. These data demonstrate that functional mitochondria are involved in hypoxia-sensing by adrenal chromaffin cells. We also show for the first time that, like their neonatal chromaffin cell counterparts, MAH cells are CO2 sensors; however, this property is independent of functional mitochondria.