Prokaryotes produce a variety of toxins that affect genomic function of both eukaryotes and prokaryotes. The 375-base pair bacterial gene Streptoalloteichus hindustanus (Sh) ble encodes a small protein, Streptoalloteichus hindustanus bleomycin resistance protein (BRP), that inhibits in vitro DNA cleavage by the prokaryotic glycopeptide bleomycin, which is a clinically used anticancer drug. NIH/3T3 cells infected with a retroviral vector containing Sh ble (SH-9 cells) were highly resistant to the cytotoxicity of bleomycin-like drugs but not to the cytotoxicity of other, structurally unrelated, DNA-cleaving agents. Expression of BRP did not markedly alter total cellular content or distribution of bleomycin-like compounds. Fluorescently labeled bleomycin was primarily localized in cytoplasmic vesicles in NIH/3T3 and SH-9 cells, whereas BRP, which has no established nuclear localization sequence, was segregated to the nucleus and more specifically to euchromatin. This karyophilic BRP may intercept bleomycin in the nucleus.