Cochllobolus heterostrophus, the fungus that causes southern corn leaf blight, was found to have a genetic system that controls heterokaryon formation between cornplementary auxotrophs. This was determined by first crossing an auxotrophic mutant with field isolates from diverse geographical locations (New York, lllinols, Mexico, and Australia) and then testing the auxotrophic progeny of each cross for ability to form heterokaryons with complementary auxotrophic tester isolates. After two generations of backcrossing to wild type, there was single-gene segregation for heterokaryon compatibility in some progenies. In other progenies there was preferential recovery of heterokaryon Incompatible spores; a possible explanation is that more than one locus controls heterokaryon compatibility (this could not be tested directly because of sexual Infertility among heterokaryon incompatible progeny). Alleles at the mating-type locus did not affect heterokaryon compatibility and segregated independently of loci controiling heterokaryon compatibility. The incompatibility system blocked the formation of heterokaryons both by hyphal anastomosis and by induced fusion of isolated protoplasts. Heterokaryon incompatibility may limit the role of heterokaryosis as a mechanism for genetic recombination in nature.