<jats:p> The antibiotic valanimycin is a naturally occurring azoxy compound produced by <jats:italic>Streptomyces viridifaciens</jats:italic> MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from <jats:sc>l</jats:sc> -valine and <jats:sc>l</jats:sc> -serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene ( <jats:italic>vlmL</jats:italic> ) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester <jats:italic>O</jats:italic> -seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway.