Daptomycin is a clinically important antibiotic approved for the treatment of complicated skin and skin structure infections caused by Gram-positive pathogens, and for the treatment of bacteremia and endocarditis caused by Staphylococcus aureus. Daptomycin and related acidic cyclic lipopeptide antibiotics have ten amino acids in the ring, and exocyclic tails containing one or three amino acids. The N-termini of the exocyclic amino acids are generally coupled to long chain fatty acids. Biosynthesis is initiated by the coupling of fatty acids to the N-terminal amino acids, followed by the coupling of the remaining amino acids by nonribosomal peptide synthetase (NRPS) mechanisms, then cyclization and release of the lipopeptides. The biosynthetic genes for daptomycin, calcium dependent antibiotic (CDA), A54145 and friulimicin have been cloned, sequenced, analyzed bioinformatically, and in some cases genetically or biochemically. The information on the organization and expression of the NRPS and other genes has been exploited to generate combinatorial libraries of hybrid lipopeptide antibiotics related to daptomycin, including several compounds with very good antibacterial activities.