Mitochondria from nitrogen-fixing soybean nodules had previously been found to display very low rates of cyanide insensitive O2 consumption and to contain little protein immunoreactive with alternative oxidase (AOX) antibodies (A. Kearns et al. 1992, Plant Physiology 99, pp. 712-717). Following the recent identification of novel regulatory mechanisms for AOX, we have re-investigated AOX activity in nodule mitochondria. When AOX is fully activated by adding pyruvate and a reductant, significant AOX activity can be measured in nodule mitochondria when substrates that cause the ubiquinone (Q) pool to become > 90% reduced are provided. These nodule mitochondria contain κ10% of the immunoreactive protein observed in soybean root or cotyledon mitochondria. Furthermore, transcripts from all three members of the soybean AOX multigene family were significantly less abundant in total RNA from nodules than from other tissues. The ratio of transcripts from the three AOX genes varied between the outer cortex and the inner infected region of the nodule. Measurements of Q redox state (Q(r)/Q(t)) versus O2 consumption showed that much higher levels of Q reduction were required for AOX to be active in mitochondria from nodules than in mitochondria from roots and cotyledons, and that root and nodule mitochondria were more dependent on added pyruvate for maximum AOX activity. Kinetic modelling suggests that these differences were simply the result of differences in the relative amount of active AOX protein, rather than differences in Q content or the AOX isoform expressed, in the different tissues.