The complete oat gene and cDNA from the commercial mushroom, Agaricus bisporus, encoding ornithine aminotransferase (OAT) was characterized. The gene encodes a 466 amino acid protein and provides the first fully reported homobasidiomycete OAT protein sequence. The gene is interrupted by ten introns, and no mitochondrial targeting motif was present pointing to a cytoplasmic localization. The function of the gene was demonstrated by complementation of a Saccharomyces cerevisiae mutant unable to utilize ornithine as a sole source of nitrogen with an A. bisporus oat cDNA construct. Northern analysis of the oat gene together with the pruA gene (encoding Delta(1)-pyrroline-5-carboxylate dehydrogenase) showed that transcripts of both genes were lower during the first stages of fruiting body development. The higher expression of the oat gene in later stages of development, suggests the importance of ornithine metabolism for the redistribution of metabolites in the developing mushroom. Hplc analysis of all amino acids revealed that ornithine levels increased during fruiting body development whereas proline levels fell.