Abstract Imatinib mesylate acts by suppressing phosphorylation of its kinase target - Bcr-Abl. We hypothesized that loss of p210Bcr-Abl (the kinase target) may lead to imatinib mesylate resistance. Two model systems were studied: K562 cells (CML blast crisis line) and MO7E/MBA-1 cells (with MBA-1 cells representing MO7E cells stably transfected with BCR-ABL). Relative resistance to imatinib mesylate developed when p210Bcr-Abl expression was abolished. Furthermore, K562 cells were significantly more growth suppressed after exposure to imatinib mesylate (p210Bcr-Abl is present but dephosphorylated), than after downregulation of Bcr-Abl expression. Signaling pathways which were functional in the absence of Bcr-Abl expression - NF-KB and MAP kinase activation or the growth factor pathway -- were disrupted when p210Bcr-Abl was present but dephosphorylated, suggesting that an intact, but enzymatically inactive Bcr-Abl, may interfere with critical growth/signaling pathways. Downregulation of p210Bcr-Abl may be a mechanism by which imatinib mesylate-resistance emerges. Samples from 3 of 15 patients with imatinib mesylate-resistant CML blast crisis had undetectable levels of p210Bcr-Abl. We conclude that retention of a dephosphorylated p210Bcr-Abl has a biologic impact distinct from that of downregulation/loss of p210Bcr-Abl and, in a subset of patients, loss of the target of the kinase inhibitor may lead to imatinib mesylate resistance.