Mice harboring the "white spotting" (W) locus have abnormalities in hematopoiesis due to one of various mutations of the c-kit proto-oncogene, which encodes the stem cell factor (SCF) receptor. The c-kit mutations identified in W mice cause diminished, absent or dominant negative receptor function. This study explores the hypothesis that acquired mutations of c-kit in the hematopoietic stem cell participate in the pathogenesis of aplastic anemia (AA). Genomic DNA was prepared from granulocytes and monocytes of 11 patients with acquired AA and one patient with a non-Fanconi's form of inherited AA. DNA was subjected to polymerase chain reaction (PCR) amplification and single-stranded conformation polymorphism (SSCP) analysis for all 21 exons of the c-kit gene. Two patients were heterozygous for a previously described polymorphism involving exon 17. Two other patient samples had an extra band on SSCP analysis of exon 10. DNA extracted from epithelial cells of one patient revealed the same SSCP pattern as that from the blood cells, suggesting that the alteration was in the germ-line. PCR-SSCP analysis of leukocyte DNA from 12 normal individuals revealed that 2 samples also had an extra band in exon 10. DNA sequencing of PCR-amplified and cloned DNA from the patients and the normal individuals with the aberrant SSCP results showed them all to be heterozygous for an ATG to CTG transition in codon 541, resulting in substitution of methionine by leucine in the transmembrane region of the protein. The same two patients and two controls were heterozygous for a silent change in codon 862 (exon 18). Matching serum samples from 4 of 6 AA patients tested had SCF levels more than two standard deviations above the normal mean value. These results suggest that neither c-kit mutations nor decreased soluble SCF levels are commonly involved in the pathogenesis of acquired AA.