The genetic suppressor element (GSE) approach allows identification of genes essential for certain cell phenotypes. To identify genes controlling the cell response to cytotoxic agents, a normalized retroviral library of randomly fragmented cDNAs from the Chinese hamster cell line DC-3F was screened for GSEs conferring resistance to bleomycin. One of these GSEs, GSE(BLM), conferring an approximately 2-fold bleomycin resistance in DC-3F cells, displayed 98% identity with an amino acid sequence located in the functional domain of human SRPK1. Using GSE(BLM) as a probe, we cloned a cDNA with a nucleotide sequence that was 76.7% identical to that of human SRPK1, whereas the corresponding amino acid sequence was 92.6% identical to that of this enzyme. When GSE(BLM), inserted in the retroviral vector pLNCX, was transduced in HeLa cells, its expression resulted in a 5-10-fold bleomycin resistance, which was abolished when these cells were further transfected with SRPK1 cDNA. In our experimental conditions, DC-3F or HeLa cells expressing GSE(BLM) did not show any detectable cross-resistance to other cytotoxic agents with various mechanisms of action. GSE(BLM), which is sense oriented in the vector, is likely to be translated in a peptide active as a dominant-negative inhibitor of SRPK1. SRPK1 is a protein serine kinase that regulates the activity of RS-proteins (arginine-serine-rich proteins), a group of nuclear factors controlling various physiological processes.