Mutations at the mouse W/c-kit locus lead to intrinsic defects in stem cells of the melanocytic, hematopoietic, and germ cell lineages. W alleles vary in the overall severity of phenotype that they confer, and some alleles exhibit an independence of pleiotropic effects. To elucidate the molecular basis for these biological differences, we analyzed the c-kit locus and the c-kit-associated autophosphorylation activities in five different W mutants representative of a range of W phenotypes. Mast cell cultures derived from mice or embryos homozygous for each W allele were deficient in c-kit autophosphorylation activity, the extent of which paralleled the severity of phenotype conferred by a given W allele both in vivo and in an in vitro mast cell coculture assay. The mildly dominant, homozygous viable alleles W44 and W57 were found to express reduced levels of an apparently normal c-kit protein. In contrast, c-kit kinase defects conferred by the moderately dominant, homozygous viable alleles W41 or W55 or the homozygous lethal allele, W37, were attributed to single-point mutations within the kinase domain of the c-kit polypeptide, which result in point substitutions of amino acid residues highly conserved in the family of protein tyrosine kinases. The nature and location of these amino acid substitutions account for the relative severity of phenotypes conferred by these W alleles and demonstrate that the pleiotropic developmental defects associated with the W/c-kit locus arise as the result of dominant loss-of-function mutations in a transmembrane receptor tyrosine kinase.