Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. Most GISTs patients respond to imatinib, yet will eventually exhibit resistance, and the mechanisms of imatinib resistance have not yet been fully elucidated. To clarify the mechanisms of secondary imatinib-resistant gastrointestinal stromal tumors, we generated resistant cells from the imatinib-sensitive GIST-T1 cells by exposing them to increasing concentrations of imatinib for 6 m. GIST-T1 IR (imatinib-resistant) cells showing an IC50 of imatinib 5-7 microM were generated. In GIST-T1 IR cells, KIT and its downstream signaling molecules remained phosphorylated with the presence of 1 microM imatinib, and no new mutations were found in KIT, PDGFRA, PKCtheta and JAK2. DNA micro-array analysis showed the overexpression of Cas-L in the resistant cells with 513 fold higher than that in the parental cells. Cas-L overexpression and SRC hyper-activation were also observed in the resistant cells at protein level and they were markedly decreased in KIT siRNA transfected GIST-T1 IR cells. Interestingly, GIST-T1 IR cells transfected with Cas-L siRNA turned out to become again sensitive to imatinib. Imatinib or PP1, a SRC inhibitor, alone was not enough to suppress the activation of KIT and its downstream signaling molecules, but the combination of them showed strong inhibitory effects on those in the resistant cells. We report for the first time that the mechanism of imatinib-resistant GISTs, at least in one cell line, involves KIT/Cas-L/SRC signaling. Cas-L depletion sensitized the resistant GIST-T1 IR cells to imatinib.