Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a β-hydroxy fatty acid with chain lengths of 13–15 carbon atoms. The lipopeptide biosurfactant was produced byBacillus subtilisOKB 105 and part of the material subjected to esterification of its Glu and Asp residues. High-resolution preparative reversed phase HPLC on EnCaPharm 100 of surfactin and its monomethyl and dimethyl esters yielded 44 fractions which were characterized by NMR and MS methods. Among the separated isoforms are the known surfactin variants withl-Leu,l-Val, orl-Ile in position 7 of the peptide ring and three hitherto unknown variants showing replacements of the leucine residues in position 2 and/or 7 byl-Val andl-Ile. Our work makes available lipoheptapeptide compounds with modified structures and different hydrophobicities which promise to have potential for biotechnological and pharmaceutical applications.