We have screened mutations of the N-ras gene at codons 12, 13, and 61 in leukemia cells obtained from 100 patients with acute myeloid leukemia (AML), and found mutated N-ras alleles in 9 patients. We further analyzed the polyclonality of multiple N-ras gene mutations in 4 AML patients. One patient, who had the monoclonal karyotype, t(11;17), had two types of double missense mutations at codons 13 and 61 in the same allele. Each of the remaining three patients, one of whom had t(15;17) with a monoclonal rearrangement of the retinoic acid receptor alpha and PML genes, carried two missense mutations in a relatively small population of leukemia cells. We have demonstrated that multiple clonality of the N-ras gene is occasionally observed in leukemia with a monoclonal karyotype. These findings indicate that the N-ras mutations may not always be characterized simply by an accumulative process and that the activated N-ras gene alone is not sufficient to cause leukemia.