The Saccharomyces cerevisiae iki mutants show an insensitive phenotype to the pGKL killer toxin, and we have cloned some IKI genes by complementation of this phenotype [Kishida et al., Biosci. Biotech. Biochem., 60, 798-801 (1996)]. Here, we identified and characterized the IKI1 and IKI3 genes. DNA sequencing of the genes showed that both have 100% identity with hypothetical genes identified by the yeast genome project, YHR187w (481,911-480,985 in chromosome VIII) for IKI1, and YLR384c (888,852-892,898 in chromosome XII) for IKI3. Both are novel genes with no significant identity with other known genes and they do not belong to any homology domain group, gene family, or superfamily. The disruption of IKI1 is not lethal, but growth of the disruptant was slower than that of the wild type at all temperatures examined. The disruptant was the killer-insensitive phenotype. The sequence of the IKI1 gene predicted a hydrophilic protein with a molecular mass of 35 kDa (309 amino acids). A 35-kDa protein band was also detected by immunoblotting the 25,000 x g pellet fraction of the wild type yeast cell lysate. Disruption of the IKI3 gene is also non-lethal and it has the killer-insensitive phenotype. Iki3p may contain a transmembrane domain near the NH2-terminal region (97-113 residues in a total of 1349 amino acids).