Oxygen consumption for bioenergetic purposes has long been thought to be the prerogative of mitochondria. Nevertheless, mitochondrial gene knockout (rho(0)) cells that are defective in mitochondrial respiration require oxygen for growth and consume oxygen at the cell surface via trans-plasma membrane electron transport (tPMET). This raises the possibility that cell surface oxygen consumption may support glycolytic energy metabolism by reoxidising cytosolic NADH to facilitate continued glycolysis. In this paper we determined the extent of cell surface oxygen consumption in a panel of 19 cancer cell lines. Non-mitochondrial (myxothiazol-resistant) oxygen consumption was demonstrated to consist of at least two components, cell surface oxygen consumption (inhibited by extracellular NADH) and basal oxygen consumption (insensitive to both myxothiazol and NADH). The extent of cell surface oxygen consumption varied considerably between parental cell lines from 1% to 80% of total oxygen consumption rates. In addition, cell surface oxygen consumption was found to be associated with low levels of superoxide production and to contribute significantly (up to 25%) to extracellular acidification in HL60rho(0) cells. In summary, cell surface oxygen consumption contributes significantly to total cellular oxygen consumption, not only in rho(0) cells but also in mitochondrially competent tumour cell lines with glycolytic metabolism.