The cluster of streptomycin (SM) production genes in Streptomyces griseus was further analysed by determining the nucleotide sequence of genes strFGHIK. The products of the strF and/or strG genes may be involved in the formation of N-methyl-L-glucosamine, and that of the strH gene in the first glycosylation step condensing streptidine-6-phosphate and dihydrostreptose. The putative StrI protein showed strong similarity to the amino-terminal NAD(P)-binding sites of many dehydrogenases, especially of the glyceraldehyde-3-phosphate dehydrogenases. The product of the strK gene strongly resembles the alkaline phosphatase of Escherichia coli. It was shown that S. griseus excretes an enzyme that specifically cleaves both SM-6-phosphate and--more slowly--SM-3''-phosphate ate during the production phase for SM. The identity of this enzyme with the StrK protein was demonstrated by expression of the strK gene in Streptomyces lividans 66. Further evidence for an involvement of these genes in SM biosynthesis came from the fact that genes homologous to them were found in the equivalent gene cluster of the hydroxy-SM producer Streptomyces glaucescens; these, however, were in part differently organized. The ca. 5 kb DNA segment downstream of strI in S. griseus which contains the strK gene was found to be located in inverse orientation between the homologues of the aphD and strR genes in S. glaucescens.