The combination of the KitW mutation and KitS allele from Mus spretus leads to male hybrid sterility. The effects of other combinations between KitW and KitM from Mus m. molossinus or KitN from Mus m. musculus on male reproductive ability were examined in this study. The KitW/KitM and KitW/KitN males were fertile and showed the normal pattern of spermatogenesis in most seminiferous tubules. There were two amino acid substitutions in the protein deduced from the cDNA sequence coded by the KitM allele sequence and three in the KitN allele compared with the protein from the + Kit allele of C57BL mice. These amino acid exchanges had no effect on the fertility of KitW/KitM and KitW/KitN males. Therefore, comparing the sequence data from cDNA coded by KitM and KitN alleles with that for the KitS allele, we concluded that one or more amino acid exchanges in the extracellular domain would be the cause of male hybrid sterility in the KitW/KitS combination; these substitutions are Phe to Ser at position 72, Thr to Ala at 95, Ser to Arg at 101, Leu to Pro at 123, and Ile to Met at 1303.