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 extra-uterine 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 (p⁰) MAH cells were hypoxia-insensitive, though responses to hypercapnia and expression of several markers, including carbonic anhydrase II (CA 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 p⁰ 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.