Ustilago hordei, the cause of barley covered smut, was found to produce a factor that inhibited its own mating. The mating inhibition factor (MIF) specifically inhibited mating of U. hordei and other Ustilago spp., but not teliospore germination or sporidial growth. MIF did prevent teliospore germination of Tilletia caries and T. contraversa. MIF was found at low levels in culture supernatants of either mating type of U. hordei grown separately, but at higher levels when both mating types were grown together, in the supernatants of MAT-1 mating type cells transformed with the MAT-1 pheromone gene mfa1 and of MAT-2 cells transformed with either mfa1 or the MAT-1 pheromone receptor gene pra1. Diploid cells produced no detectable inhibitor, nor did MAT-1 cells with a disrupted mating type locus that deleted both mfa1 and pra1. MIF production was restored when mfa1, but not pra1, was added back to the MAT-1Δ cells. MIF activity was altered by protease treatment. Highly purified MIF from MAT-1 cells contained cysteine methyl ester, farnesyl cysteine, farnesyl cysteine methyl ester, and a dodecapeptide with a mass consistent with that of MAT-1 pheromone lacking the terminal cysteine. Since smut fungi must first mate to become pathogenic, mating inhibition has the potential to be an effective method of disease control for these pathogens.