<jats:p>The nucleotide sequence of the streptothricin acetyltransferase (STAT) gene from streptothricin-producing Streptomyces lavendulae predicts a 189-amino-acid protein of molecular weight 20,000, which is consistent with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. The amino acid composition and the NH2-terminal sequence determined by using the purified protein are in good agreement with those predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. High-resolution S1 nuclease protection mapping suggests that transcription initiates at or near the adenine residue which is the first position of the translational initiation triplet (AUG) of STAT. Another open reading frame located just upstream of the STAT gene was detected and contains a region bearing a strong resemblance to DNA-binding domains which are conserved in known DNA-binding proteins. By addition of promoter signals and a synthetic ribosome-binding (Shine-Dalgarno) sequence at an appropriate position upstream of the STAT translational start codon, the STAT gene confers streptothricin resistance on Escherichia coli and Bacillus subtilis. The STAT coding sequence with both the promoter of a B. subtilis cellulase gene and a synthetic Shine-Dalgarno sequence was functionally expressed in Streptomyces lividans, which suggests that the addition of an artificial leader upstream of the translational initiation codon (AUG) does not significantly influence the translation of STAT.