Formation of the hybrid BCR-ABL gene is responsible for >95% of chronic myeloid leukemia (CML). The alternative, downstream ABL promoter (Pa), which is usually retained in this chimeric oncogene, was reported to be methylated in many CML patients, but there has been controversy as to whether this methylation is a frequent change in bone marrow (BM) in early chronic phase (CP) or only past this stage. Also, the relevance of Pa promoter methylation to BCR-ABL expression in CML is unclear. We examined methylation of the ABL Pa promoter in uncultured BM samples and in colonies derived from their hematopoietic precursor cells by bisulfite and PCR-based assays (combined bisulfite restriction analysis and methylation-specific PCR). BM from seven CP CML patients at diagnosis had about 20-60% of the copies of the ABL Pa promoter methylated. No Pa methylation was detected in normal BMs or colonies derived from them. In contrast, most colonies from CP CML patients had Pa methylation. Surprisingly, 18-49% of the CML-derived colonies with this methylation reproducibly had no detectable BCR-ABL RNA on nested reverse transcription-PCR. Furthermore, the percentage of BCR-ABL RNA-positive colonies was almost same among the colonies not displaying Pa methylation as among the colonies in which this methylation was found. We conclude that ABL Pa methylation is often an early marker of CML in hematopoietic precursors and in total mononuclear BM cells but that it is not associated with an increased frequency of BCR-ABL RNA-positive cells. This methylation might be emblematic of cancer-associated hypermethylation elsewhere in the genome with the consequent silencing of tumor suppressor genes seen in many malignancies.