The major mechanism of action of STI571 is a competitive interference with the ATP-binding site of the Bcr/Abl tyrosine kinase. In the BCR/ABL-positive cell line KBM5, we studied cellular events associated with the in vitro acquisition of resistance to STI571. The emergence of the STI571-resistant phenotype was accompanied by only a marginal increase in the number of copies of the BCR/ABL gene and its level of expression. The activity of the Bcr/Abl kinase (level of autophosphorylation) in resistant cells was, however, incompletely inhibited by STI571, and the acquisition of the high degree of resistance was associated with a single-point mutation leading to a substitution of a threonine-to-isoleucine at position 315 of Abl. In the resistant KBM5-STI571^R1.0 cells, 20% of the BCR/ABL transcripts and 10% of BCR/ABL gene copies on the DNA level were mutated. The mutation was present in all 10 STI571-resistant clones derived from low density clonogenic assay, confirming its presence in all colony-forming cells but only in a fraction of the BCR/ABL gene copies in each cell. The contribution of this mutation to STI571-resistant phenotype remains unknown. Preliminary data showing partial reversibility of resistance in these cells suggest that resistance may be multifactorial. No other mutations were identified in the kinase domain of the BCR/ABL gene.