The Tri4 gene of Fusarium sporotrichioides was isolated from a cloned DNA fragment carrying the Tri5 gene by complementation of a Tri4- mutant. The nucleotide sequence of Tri4 was determined and the locations of three introns were identified. Analysis of Tri4 mRNA levels revealed that transcription reached maximum levels coincidently with the onset of trichothecene biosynthesis, and then declined 20-fold over the next 8 h. Disruption of Tri4 resulted in the loss of production of both trichothecenes and apotrichodiol and the accumulation of the unoxygenated pathway intermediate trichodiene. Transformants lacking a functional Tri4 gene were able to convert isotrichotriol, an early pathway intermediate, to T-2 toxin suggesting that most pathway enzymes are present in Tri4- mutants. These data suggest that the enzyme encoded by Tri4 catalyzes the first oxygenation step in the trichothecene pathway and participates in apotrichodiol biosynthesis. Tri4 encodes a protein of 520 residues (M(r) = 59 056) that shows significant homology with members of the superfamily of cytochromes P450. It appears most similar to the CYP3A subfamily (24.6% amino acid identity). Because it contains less than 40% positional identity with other cytochromes P450, the Tri4 gene has been placed in a new cytochrome P450 gene family designated CY P58.