To improve the curative success of chemotherapy, it will be essential to understand the molecular basis of drug resistance (DR) and sensitivity. We have developed a cell culture system that enables the functional cloning of mammalian DR genes based on phenotypic selection after overexpression of mammalian retroviral cDNA libraries and validated our system using the anticancer drug cisplatin. ERCC1-deficient and therefore cisplatin-hypersensitive mouse embryonic fibroblast target cells were transduced with a human placenta retroviral cDNA library. Subsequent cisplatin selection yielded 20 DR clones, each containing a recurring human ERCC1 gene. Surprisingly, nine of these clones contained 5'-truncated ERCC1 sequences that required alternative splicing of the vector sequence to encode a functional ERCC1 protein. The usage of cryptic splice sites in the vector sequence should be taken into consideration when interpreting results from retroviral gene expression applications, and might have consequences for the safe application of retroviral constructs in gene therapy. (C) 2003 Elsevier Inc. All rights reserved.