The cyclic tetrapeptide HC-toxin is required for pathogenicity of the filamentous fungus Cochliobolus carbonum on maize. HC-toxin production is controlled by a complex locus, TOX2. The isolation and characterization of a new gene of the TOX2 locus, TOXF, is reported. It is shown that TOXF is specifically required for HC-toxin production and pathogenicity. It is present as two or three copies in all HC-toxin-producing (Tox2+) isolates and is absent in toxin-non-producing strains. The deduced amino acid sequence of TOXF has moderate homology to many known or putative branched-chain-amino-acid transaminases from various species. A strain of C. carbonum with all copies of TOXF disrupted grew normally but lost HC-toxin production and pathogenicity. It is proposed that TOXF has a biosynthetic role in HC-toxin synthesis, perhaps to aminate a precursor of Aeo (2-amino-9,10-epoxi-8-oxodecanoic acid).