The nucleotide sequence of an aminoglycoside phosphotransferase gene (rph) from Streptomyces ribosidificus (a ribostamycin producer) was determined. Molecular size, amino acid composition and N-terminal amino acid sequence of the purified rph product confirmed the position of the coding region deduced from the nucleotide sequence. The 5' region of rph has been tested for its transcriptional controls; high-resolution mung-bean nuclease mapping of in vivo transcripts revealed one major start point, rphS1, controlled by the rphP1 promoter. This transcript was also observed in vitro in run-off experiments using purified Streptomyces RNA polymerase. This transcriptional start point coincided with the translational start site, with the mRNA 5' terminus being pppATG. The results of promoter-probing tests and insertion of a transcriptional termination fragment into the rph promoter region have shown that the rphP1 transcript was sufficient and essential for rph expression.