We determined the proportion of oxygen consumption due to oxidative phosphorylation by mitochondria in an adrenal gland cell line (Y1 cells). In addition we determined the relative proportion of in situ mitochondrial oxygen consumption attributable to (i) proton leak and (ii) ATP turnover in these cells. This approach allowed use of top-down elasticity analysis to determine control of oxidative phosphorylation by mitochondrial (a) proton leak flux (b) substrate oxidation flux and (c) ATP turnover flux, as a function of changes in in situ mitochondrial membrane potential. Our data show that resting oxygen consumptions rates of Y1 cells to be 87 +/- 7 nmolO/min/10(7) cells of which 38 +/- 3% was not due to oxidative phosphorylation. We demonstrated that mitochondrial proton leak accounted for 7 +/- 3% of total cellular oxygen consumption or 12 +/- 6% of resting mitochondrial oxygen consumption, with ATP turnover accounting for 55 +/- 3% of total cellular oxygen consumption or 78 +/- 6% of mitochondrial oxygen consumption. Control of resting mitochondrial oxygen consumption in Y1 cells was shared by (a) substrate oxidation flux (37 +/- 8%), (b) proton leak flux (15 +/- 8%) and (c) ATP turnover (56 +/- 8%). Our data demonstrate, for the first time, that the majority of oxygen consumption by resting Y1 cells is due to oxidative phosphorylation.