Alternative oxidase (AOX) is a mitochondrial protein that acts as an alternative terminal oxidase to the conventional cytochrome oxidases. It is present in certain prokaryotes, plants, fungi and some protozoa but absent in mammals. AOX activity has previously been described in Acanthamoeba, although no genetic evidence has been reported. Herein, two AOX (AcAOX) genes designated isoforms A and B, were obtained from Acanthamoeba castellanii by a combination of degenerate PCR from cDNA and a series of 5' and 3' rapid amplification of cDNA ends. The corresponding genomic sequences of these AcAOXs were also obtained. Each gene spans six exons over a region of 1607 and 1619bp, respectively. Isoforms A and B have open reading frames of 1113 and 1125bp, respectively. Each encodes a protein with a predicted molecular weight of 42kDa. Each AcAOX protein has a predicted cleavable mitochondrial targeting sequence. The full-length AcAOX is functionally active as it complements hemL-deficient Escherichia coli and inhibited by the inhibitor of AOX, salicylhydroxamic acid (SHAM). SHAM is effective against A. castellanii and Acanthamoeba polyphaga only when used in conjunction with antimycin A, an inhibitor of the conventional cytochrome respiratory pathway. Transcripts for AcAOX are increased during the encystment process, indicating a possible role for alternative respiration during stress.