ABSTRACT The tangerine pathotype of Alternaria alternata produces a host-selective toxin (HST), known as ACT-toxin, and causes Alternaria brown spot disease of citrus. The structure of ACT-toxin is closely related to AK- and AF-toxins, which are HSTs produced by the Japanese pear and strawberry pathotypes of A. alternata, respectively. AC-, AK-, and AF-toxins are chemically similar and share a 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Two genes controlling AK-toxin biosynthesis (AKT1 and AKT2) were recently cloned from the Japanese pear pathotype of A. alternata. Portions of these genes were used as heterologous probes in Southern blots, that detected homologs in 13 isolates of A. alternata tangerine pathotype from Minneola tangelo in Florida. Partial sequencing of the homologs in one of these isolates demonstrated high sequence similarity to AKT1 (89.8%) and to AKT2 (90.7%). AKT homologs were not detected in nine isolates of A. alternata from rough lemon, six isolates of nonpathogenic A. alternata, and one isolate of A. citri that causes citrus black rot. The presence of homologs in the Minneola isolates and not in the rough lemon isolates, nonpathogens or black rot isolates, correlates perfectly to pathogenicity on Iyo tangerine and ACT-toxin production. Functionality of the homologs was demonstrated by detection of transcripts using reverse transcription-polymerase chain reaction (RT-PCR) in total RNA of the tangerine pathotype of A. alternata. The high sequence similarity of AKT and AKT homologs in the tangerine patho-type, combined with the structural similarity of AK-toxin and ACT-toxin, may indicate that these homologs are involved in the biosynthesis of the decatrienoic acid moiety of ACT-toxin.