The identity of the multiple alternative oxidase bands detected in various soybean tissues was investigated to determine if any modification that can alter the mobility on SDS-PAGE of the alternative oxidase occurs after mitochondrial import other than removal of the presequence. Comparison of the mature, in vitro imported products of AOX1, AOX2 and AOX3 in soybean cotyledons and rat liver mitochondria indicated that they had an identical apparent molecular mass to their in vitro expressed mature forms. This suggests that no modification specific to plant alternative oxidase altering the mobility on SDS-PAGE, took place. Changing the –2 and/or –3 Arg residue resulted in the inhibition of the generation of this mature form, suggesting that processing was most likely by the general mitochondrial processing peptidase. Comparison of the in vitro expressed mature forms to that detected by immunoblots of soybean tissues, required the induction of AOX1. Treatment of soybean cultured cells with antimycin A resulted in the induction of an additional band cross-reacting to monoclonal antibodies against the alternative oxidase. Comparison of the in vitro expressed mature forms to the alternative oxidase detected by western blotting indicated that they were identical in apparent molecular mass. These results indicated that no modification other than presequence removal, which alters mobility on SDS-PAGE, was required to generate the mature functional alternative oxidase proteins.