Mutations in the ras proto-oncogenes are the most frequently observed molecular alteration in acute myeloid leukemia (AML). Whether ras mutations occur as late or relatively early events in the multistep process of myeloid transformation, remains an open question. We previously described illegitimate T-cell receptor (TCR)-delta gene rearrangements in a subset of AML. These recombinations were detected in 9 out of 100 de novo AML cases. Southern blot analysis suggested the presence of these recombinations in the vast majority of AML cells and thus could be used as clonal markers. In order to more accurately define the role of ras proto-oncogene mutations in the multistep process of malignant transformation in myeloid leukemias, we performed single strand conformation polymorphism (SSCP) assays, slot blot and direct sequencing analysis on these nine cases with illegitimate TCR delta gene rearrangements. Ras proto-oncogene mutations were found in three of nine cases. Interestingly, SSCP, slot blot and sequencing suggested the presence of the respective mutations in most of the leukemic cells. Thus, ras mutations presumably occurred early in the process of transformation in these three cases.