Bcl-x(L), a prosurvival member of the Bcl-2 family that is expressed in many tumors, represses apoptosis induced by chemotherapeutic drugs in vitro. However, the contribution of apoptosis and prosurvival Bcl-2-related proteins to chemotherapy resistance in vivo is unknown and has been challenged by recent results with clonogenic survival assays. To test the ability of Bcl-x(L) to provide chemotherapy resistance to tumors, we transfected the mouse bcl-x(L) gene into the tumorigenic SCK mammary cell line and assessed the response of tumor cells to chemotherapeutic drugs in clonogenic assays and in a syngeneic mouse model. Bcl-x(L) conferred protection on SCK cells against methotrexate at certain drug concentrations, but not at all against 5-fluorouracil in clonogenic survival assays in vitro. Injection of SCK cells transfected with Bcl-x(L) or control plasmid in the mammary fat pads of syngeneic recipient mice resulted in tumors of similar size. However, although the volume of control tumors regressed up to 80% after 4 to 5 days of chemotherapy, SCK tumors expressing Bcl-x(L) did not regress and continued to grow in the presence of methotrexate or 5-fluorouracil. In addition, numbers of apoptotic cells were significantly higher in control tumors as compared to Bcl-x(L)-expressing tumors in animals treated with methotrexate or 5-fluorouracil. These results provide evidence that inhibition of apoptosis through Bcl-x(L) overexpression can promote resistance to chemotherapy in tumors in vivo.