<jats:p>Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism in<jats:italic>Streptomyces roseosporus</jats:italic>. A 128 kb region of<jats:italic>S. roseosporus</jats:italic>DNA was cloned and verified by heterologous expression in<jats:italic>Streptomyces lividans</jats:italic>to contain the daptomycin biosynthetic gene cluster (<jats:italic>dpt</jats:italic>). The cloned region was completely sequenced and three genes (<jats:italic>dptA</jats:italic>,<jats:italic>dptBC</jats:italic>,<jats:italic>dptD</jats:italic>) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acid<jats:sc>l</jats:sc>-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (<jats:italic>dptE</jats:italic>,<jats:italic>dptF</jats:italic>) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (<jats:italic>dptI</jats:italic>,<jats:italic>dptJ</jats:italic>) probably involved in supply of the non-proteinogenic amino acids<jats:sc>l</jats:sc>-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would have<jats:sc>d</jats:sc>-Asn, rather than<jats:sc>l</jats:sc>-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.