The Japanese pear pathotype of Alternaria alternata produces host-specific AK-toxin and causes black spot of Japanese pear. Previously, a cosmid clone, pcAKT-1, was isolated that contains two genes, AKT1 and AKT2, within a 5.0-kb region required for AK-toxin biosynthesis. The wild-type strain has multiple, nonfunctional copies of these genes. In the present study, two additional genes, AKTR-1 and AKT3-1, downstream of AKT2 were identified. Transformation of the wild type with AKTR-1- and AKT3-1-targeting vectors produced toxin-deficient (Tox-), nonpathogenic mutants. DNA gel blot analysis, however, demonstrated that the fragments targeted in Tox- mutants were different from those containing AKTR-1 and AKT3-1 on the transforming vectors. A cosmid clone, pcAKT-2, containing the targeted DNA was isolated and shown to carry two genes, AKTR-2 and AKT3-2, with high similarity to AKTR-1 and AKT3-1, respectively. Transcripts from not only AKTR-2 and AKT3-2 but also AKTR-1 and AKT3-1 were found in the wild type. DNA gel blot analysis with pulsed-field gel electrophoresis showed that AKT1, AKT2, AKT3, and AKTR and their homologues are on a single chromosome. These results indicate the structural and functional complexity of the genomic region controlling AK-toxin biosynthesis.