The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not apparent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respiratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regulation of respiratory flux via the alternative pathway.