A 3.2 kb Sstl-Sphl DNA fragment of Streptomyces antibioticus, an oleandomycin producer, conferring resistance to oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its deduced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC-transporter superfamily including the gene product of another oleandomycin-resistance gene (OleC). The OleB protein contains two ATP-binding domains, each of approximately 200 amino acids in length, and no hydrophobic transmembrane regions. Functional analysis of the oleB gene was carried out by deleting specific regions of the gene and assaying for oleandomycin resistance. These experiments showed that either the first or the second half of the gene containing only one ATP-binding domain was sufficient to confer resistance to oleandomycin. The gene oleB was expressed in Escherichia coli fused to a maltose-binding protein (MBP) using the pMal-c2 vector. The MBP-OleB hybrid protein was purified by affinity chromatography on an amylose resin and polyclonal antibodies were raised against the fusion protein. These were used to monitor the biosynthesis and physical location of OleB during growth. By Western analysis, the OleB protein was detected both in the soluble and in the membrane fraction and its synthesis paralleled oleandomycin biosynthesis. It was also shown that a Streptomyces albus strain, containing both a glycosyltransferase (OleD) able to inactivate oleandomycin and the OleB protein, was capable of glycosylating oleandomycin and secreting the inactive glycosylated molecule. It is proposed that OleB constitutes the secretion system by which oleandomycin or its inactive glycosylated form could be secreted by S. antibioticus.