We screened for natural mutations in Crl:CF1 closed colony mice using an ordinary backcrossing system. Five of 30 CF1 males carried novel genes that caused white spots on colored coats. Their backcross progenies showed a white spot phenotype. The white spot gene was mapped to approximately 39 cM on chromosome 5, where the Kit gene is known to reside. Allelism testing between this spot gene and the Kit gene was performed using two already known Kit alleles, Kit(W), and Kit(W-v). We demonstrated that the spot mutation was semidominant and a novel allele of the Kit gene, which was tentatively named Kit(W-Ham). No infertility or anemia was observed in Kit(W-Ham) homozygotes. However, a reduced number of germ cells and mast cells was observed in Kit(W-Ham)/Kit(W) and Kit(W-Ham)/Kit(W-v) transheterozygotes. Sequencing of the 21 exons of the Kit gene in the Kit(W-Ham) mutants revealed that a unique guanine-to-adenine (G-A) transition at nucleotide position 545 (c.545G>A) of exon 3 changes arginine (R) to glutamine (Q) at position 182 in the extracellular domain of the KIT protein (p.R182Q). This extracellular KIT domain is a binding site for stem cell factors (SCF). It was concluded that the Kit(W-Ham) mutant may serve as a new model of human piebaldism.