ABSTRACT The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerines and tangerine hybrids. Sequence analysis of a genomic BAC clone identified a previously uncharacterized portion of the ACT-toxin biosynthesis gene cluster (ACTT). A 1,034-bp gene encoding a putative enoyl-reductase was identified by using rapid amplification of cDNA ends and polymerase chain reaction and designated ACTTS2. Genomic Southern blots demonstrated that ACTTS2 is present only in ACT-toxin producers and is carried on a 1.9 Mb conditionally dispensable chromosome by the tangerine pathotype. Targeted gene disruption of ACTTS2 led to a reduction in ACT-toxin production and pathogenicity, and transcriptional knockdown of ACTTS2 using RNA silencing resulted in complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS2 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.