AbstractDespite the absence of an alternative mitochondrial ubiquinol oxidase, Saccharomyces cerevisiae consumes oxygen in an antimycin A‐ and cyanide‐resistant manner. Cyanide‐resistant respiration is typically used when the classical respiratory chain is impaired or absent (i.e in anaerobically‐grown cells shifted to normoxia or in respiratory‐deficient cells). We characterized the non‐respiratory oxygen consumption pathways operating during anoxic–normoxic transitions in glucose‐repressed resting cells. High‐resolution oxygraphy confirmed that the cellular non‐respiratory oxygen consumption pathway is sensitive to high concentrations of cyanide, azide, SHAM and TTFA, and revealed several new characteristics. First, the use of sterol biosynthesis inhibitors showed that this pathway makes a considerable contribution (about 25%) to both endogenous and glucose‐dependent oxygen consumption. Anaerobically‐grown glucose‐repressed cells exhibited high apparent oxygen affinities (Km for oxygen = 0.5–1 µM), even in mutants deficient in respiration or sterol synthesis. Exogeneously added glucose and endogenous stored carbohydrates were the only substrates that were efficient for cellular oxygen consumption (apparent Km for exogenous glucose = 2–3 mM). On the other hand, fluorimetric measurements of the cellular NAD(P)H pool showed that the cellular oxygen consumption (sterol biosynthesis and unknown pathways) was dependent more on the intracellular level of NADPH than of NADH. High oxygen affinity NADPH‐dependent oxygen consumption systems were thought to be mainly localized in microsomal membranes, and several data indicated a significant contribution made by uncoupled P450 systems, together with still uncharacterized systems. Such activities are associated in vitro with a massive production of O2·− and, to a lower extent, H2O2 and a likely concomitant production of H2O. Copyright © 2002 John Wiley & Sons, Ltd.