Wepreviously reported that a 7.6-kb DNAfragment from Streptomyces kasugaensis M338-M1 , a kasugamycin (KSM) producer, included KSMacetyltransferase gene (kac33S) and some other genes possibly involved in KSMbiosynthesis. As an extension of that study, a 10-kb Sacl-Kpnl DNAfragment, located 5~15-kb upstream of kac?3%, was cloned and a 4.2-kb Sacl-EcoRl fragment there from was sequenced, revealing one incomplete (designated ORFJ) and three complete open reading frames (designated kasK, kasL and kasM). The coding frames of kasK, L and Moverlap one another with terminator/initiator ATGAsequence. RT-PCRanalysis of a DNAregion including kasKLMindicated the presence of one transcript that is long enough to span the three genes. The kasK gene potentially encodes an ATP-binding protein of the ATPbinding cassette (ABC) transporter super family. Homology search for the deduced KasK protein shows similarity to other ABCtransporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain. The kasL and kasM genes encode different integral membraneproteins, both having six putative transmembrane helices. Anexpression plasmid for kasKLM(pTV^KLM) was constructed and these genes were expressed in E. coli JM109, which had been sensitive to KSM.The transformant acquired resistance to KSM,suggesting that KasK, L and Mproteins as a set in S. kasugaensis M338-M1pump out KSMto protect the producer from its toxic metabolite. Organisms producing potentially autotoxic antibiotics possess basically three types of self-resistance mechanisms to avoid suicide: (1) modification of the target site that the antibiotic acts on, (2) intracellular inactivation of the antibiotic and (3) exclusion of the antibiotic from the cell1T he last resistance mechanism is designated "membraneassociated system" consisting of two classes. In one class, resistance is mediated by membrane proteins, which are believed to energize export of antibiotic molecules by proton-dependent transmembrane electrochemical gradients. The other class belongs to the ABCtransporter superfamily2) comprising many membrane-associated export and import systems, which are present both in prokaryotic and in eukaryotic cells. ABC transporters