Based on analyses of two-dimensional gel and cDNA microarrays, our laboratory and others have demonstrated that a number of genes show altered expression during the development of cisplatin resistance (CP-r) in human cancer cells, including genes associated with DNA damage repair, proto-oncogenes, apoptosis, stress-response, and transcription factors. To verify these results and find genes that are directly responsible for CP-r, as opposed to those reflecting a secondary response induced by cisplatin treatment or resulting from CP-r, we constructed a retroviral cDNA library in the vector pLNCX2 from KB-CP.5 (KCP.5), a cell line selected in one step after exposure to cisplatin at 0.5 microg/ml. Using a library of cDNAs (1.8 x 10(6) cDNA clones) and an intermittent cisplatin selection system to allow more effective functional cloning, 11 expressed cDNAs were identified in a primary pool of 93,000 transfected cell clones. Metallothionein 2A, a known CP-r gene, was among these 11 genes found in the transfectants after CP selection. Several other genes, including those encoding ribosomal proteins (e.g., RPL36) and heat shock protein (e.g., HSP10), were also found among the cisplatin-selected clones. Transfection of either the RPL36 cDNA or HSP10 cDNA conferred on KB-3-1 cells 2.5- to 3-fold resistance to cisplatin by clonogenic assays. A subsequent transfection also identified RPL36 as a CP-r gene. The finding that a ribosomal protein gene, RPL36, contributes to CP-r should stimulate study of the role of ribosomal proteins in multifactorial mechanisms of cisplatin resistance.