Adriamycin is one of the most active anticancer drugs but the development of resistance to this drug hampers its efficacy. In an effort to identify novel genes that confer resistance to adriamycin, we introduced a yeast genomic library into Saccharomyces cerevisiae and selected transformants that grew in the presence of a normally toxic concentration of adriamycin. Detailed examination of a plasmid recovered from these transformants revealed that overexpression of the gene for Ssl2p rendered yeast cells resistant to adriamycin. Ssl2p is a protein that is involved in the initiation of transcription and in DNA repair. Overexpression of Ssl2p did not confer resistance to aclarubicin, an anthracycline anticancer drug, which, like adriamycin, is intercalated into DNA. Both adriamycin and aclarubicin inhibit topoisomerase II and, thus, topoisomerase II might not be a major factor in the acquired resistance to adriamycin that results from overexpression of Ssl2p. We tested several other compounds but the only one to which Ssl2p-over-expressing cells were cross-resistant was actinomycin D. Mammalian cells that overexpress P-glycoprotein, which is a transmembrane protein that is involved in the efflux of certain drugs, are resistant to both adriamycin and actinomycin D but not to aclarubicin. However, overexpression of Ssl2p had little or no effect on the intracellular accumulation of adriamycin. Our results suggest that a novel mechanism might be involved in the sensitivity of yeast to both adriamycin and actinomycin D. (C) 2003 Elsevier Inc. All rights reserved.