Alternative oxidase activity (oxygen uptake in the presence of KCN, antimycin or myxothiazol) in mitochondria isolated from the roots of soybean seedlings was very slow, even with succinate as substrate. This activity was stimulated substantially (100-400%) by the addition of pyruvate, with half maximal stimulation occurring at 0.1 mM pyruvate. Mitochondria from soybean shoots displayed high alternative oxidase activity with succinate and malate as substrates but lower activity with exogenous NADH; addition of pyruvate stimulated the activity with NADH up to that seen with succinate. This stimulation of cyanide-insensitive NADH oxidation was seen also with mitochondria from other species. Hydroxypyruvate and oxoglutarate could substitute for pyruvate, although higher concentrations were required to achieve maximum stimulation. Pyruvate stimulation of cyanide-insensitive oxygen uptake was observed with exogenous quinols as substrates, with sub-mitochondrial particles, and in the presence of the pyruvate transport inhibitor, cyanohydroxycinnamic acid, but was not observed with detergent-solubilised mitochondria. It is suggested that pyruvate acts allosterically on the alternative oxidase to stimulate its activity. The implications of these findings for respiration in vivo are discussed.