A central segment of a cluster of biosynthetic genes for the antibiotic streptomycin cloned from Streptomyces griseus was analysed for open reading frames, as well as for transcriptional and translational activity. The nucleotide sequence revealed two significant open reading frames, ORF1 and APH(6), orientated in opposite directions and with a spacer of 885 bp between the start codons. The first, ORF1, had a coding capacity of 38 kDa. One open reading frame, APH(6), was identified as the major resistance gene coding for streptomycin 6-phosphotransferase, a protein of 307 amino acid residues and 33 kDa. Sequence determination of the first 14 N-terminal amino acid residues of the purified APH(6) enzyme protein was in agreement with the proposed primary structure. The possible identity of the presumed gene product of ORF1 with an in vitro translated protein (apparent molecular weight 41 kDa) is discussed. Comparison of the two APH(6) genes from S. griseus and the hydroxystreptomycin-producing S. glaucescens (cf. Vogtli and Hutter 1987) revealed 75% nucleotide sequence homology in the coding region and 74% conservation of the polypeptide sequence. Two protein domains which are highly conserved in other antibiotic and protein phosphotransferases were detected.