Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive. Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants. A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity. The temperaturesensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin. These results led to the reexamination of other mutants that were known to complement in vitro. Each was found to secrete one of the two polypeptides. Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect. Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides. Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction. Also, the two polypeptides do not share common antigenic determinants. The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect. The similarity of the U. maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed.