Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.