Two Cys residues, Cys(I) and Cys(II), are present in most plant alternative oxidases (AOXs). Cys(I) inactivates AOX by forming a disulfide bond with the corresponding Cys(I) residue on the adjacent subunit of the AOX homodimer. When reduced, Cys(I) associates with alpha-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the Cys(I) site. Cys(II) may also be a site of AOX activity regulation, through interaction with the small alpha-keto acid, glyoxylate. Comparison of Arabidopsis AOX1a (AtAOX1a) mutants with single or double substitutions at Cys(I) and Cys(II) confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at Cys(I)) was limited to Cys(I). A variety of Cys(II) substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at Cys(I), charge repulsion is involved. Independent effects at each Cys were suggested by lack of Cys(II) substitution interference with pyruvate stimulation at Cys(I), and close to additive activation at the two sites. However, results obtained using diamide treatment to covalently link the AtAOX1a subunits by the disulfide bond indicated that Cys(I) must be in the reduced state for activation at Cys(II) to occur.