The copy number of the alternative oxidase gene, Aox, was investigated in soybean (Glycine max L.) using a Polymerase chain reaction (PCR) approach to amplify fragments from soybean genomic DNA. The primers used were based on absolutely conserved regions of Aox cDNA clones from a variety of plant species and the yeast Hansenula anomala. After subcloning of the 170-bp PCR products, 12 individual colonies were sequenced. Eleven plasmids yielded inserts representing three sequences in the ratio 4:3:4 (Aox1-3). The sequence of Aox1 was 100% identical at the nucleic acid level to the published full-length cDNA from soybean. The other two sequences were 60-75% identical to Aox1 and to each other at the nucleic acid and protein levels. Similar analysis of Nicotiana tabacum L. revealed an additional gene copy with high homology to the soybean Aox2 sequence. Genomic DNA from soybean cut with Hind III and probed with the full-length Aox1 yielded a single positive band of 6.5 kb; when the same genomic blot was probed with a mixture of all three PCR fragments, three bands of 9 kb, 6.5 kb and 3 kb were detected. Reverse transcription-PCR performed on total RNA from various soybean tissues, followed by hybridisation with the three Aox sequences individually, revealed differential expression of the Aox genes between cotyledons and leaves. It is suggested that soybean contains a multigene Aox family. The implication of this for the understanding of alternative oxidase expression and regulation in plant tissues is discussed.