Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.