Mating type (MAT) genes were cloned from three members of the Gibberella/Fusarium complex that differ in reproductive mode: heterothallic G. fujikuroi, homothallic G. zeae, and asexual F. oxysporum. The G. fujikuroi MAT locus organization is typical of other heterothallic pyrenomycetes characterized to date; i.e., there are three genes at MAT1-1 and one at MAT1-2. G. zeae has homologues of all four genes encoded by the two G. fujikuroi MAT idiomorphs, tightly linked on the same chromosome, interspersed with sequences unique to G. zeae. Field isolates of F. oxysporum, although asexual, have either the MAT1-1 or the MAT1-2 genes found in sexual species and these genes are highly similar to those of heterothallic G. fujikuroi. RT-PCR analysis proved that the F. oxysporum MAT genes are expressed and that all putative introns found in each of the four MAT genes in G. fujikuroi and F. oxysporum are removed. Apparent failure of F. oxysporum to reproduce sexually could not be attributed to mutations in the MAT genes themselves.