Activity of the plant mitochondrial alternative oxidase (AOX) can be regulated by organic acids, notably pyruvate. To date, only two well-conserved cysteine residues have been implicated in this process. We report the functional expression of two AOX isozymes (Sauromatum guttatum Sg-AOX and Arabidopsis thaliana At-AOX1a) in Schizosaccharomyces pombe. Comparison of the response of these two isozymes to pyruvate in isolated yeast mitochondria and disrupted mitochondrial membranes reveals that in contrast to At-AOX1a, Sg-AOX activity is insensitive to pyruvate and appears to be in a constitutively active state. As both of these isozymes conserve the two cysteines, we propose that such contrasting behaviour must be a direct result of differences in their amino acid sequence and have subsequently identified novel candidate residues.