We have investigated the involvement of the p53 tumor suppressor gene and RAS family proto-oncogenes in BCR/ABL-negative chronic myeloproliferative disorders (CMPD), including nine cases of myelosclerosis with myeloid metaplasia, four polycythemia vera, 10 essential thrombocythemia, one juvenile chronic myeloid leukemia, and eight BCR/ABL-negative chronic myeloid leukemia. Twenty-five samples were studied in the chronic phase, while seven samples were analyzed in the acute accelerated or blastic phase. The presence of mutations in p53 exons 5-9, as well as in N-, K-, H-Ras exons 1 and 2 (containing codons 12, 13, and 61) was tested by the polymerase chain reaction (PCR) single strand conformation polymorphism technique and by PCR direct sequencing. In addition, restriction analysis was performed to screen for gross rearrangements within the p53 locus. Alterations of the p53 tumor suppressor gene and Ras family proto-oncogenes were detected in 2/7 and 3/7 cases of acute phase BCR/ABL-negative CMPD, respectively, while consistently negative in all the chronic phase samples analyzed. These results suggest that p53 inactivation and/or Ras activation might play a role in acute transformation of BCR/ABL-negative CMPD.