Mating and pathogenesis of the corn smut fungus, Ustilago maydis, are controlled by two unlinked mating-type loci, a and b. Yeast-like haploids that differ at both loci are compatible and fuse to establish a pathogenic dikaryon. Mating is assayed in vitro by co-inoculation on culture medium containing activated charcoal; compatible combinations have a characteristic "fuzzy" appearance caused by the growth of aerial hyphae. In general, this test has not been useful for assaying the mating ability of strains that are already mycelial (e.g., those heterozygous at b or at both mating-type loci). Using an assay for cytoduction involving transfer of a mitochondrial marker during transient cell fusion, and engineered strains with defined genotypes, we examined the mating abilities of strains heterozygous or hemizygous at the mating-type loci. The data (which have not been available from conventional pathogenicity or plate mating tests) show that heterozygosity at b attenuates fusion in haploid and diploid strains, whereas strains heterozygous at a retain the ability to fuse with a compatible haploid partner. It appears, therefore, that subsequent fusion events are attenuated once fusion has occurred to establish the U. maydis dikaryon.