The spectinomycin (sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 1 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.