<jats:title>Abstract</jats:title><jats:p>From a submerse culture of the fungus <jats:italic>Hypocrea muroiana</jats:italic> Hino et Katsumoto (Ascomycetes: Hypocreales) two major groups of peptides, designated hypomurocin (HM) A and B, belonging to the peptaibol family, could be characterized. Both groups showed antibiotic activity (<jats:italic>Bacillus subtilis</jats:italic>) and caused hemolysis of rat erythrocytes, HM A being less active than HM B. Peptides were isolated from the culture broth by chromatography on XAD‐2 adsorber resin, octyl silica, and Sephadex LH 20. HM A and B were separated by preparative TLC, and individual peptides from each microheterogeneous group were isolated by preparative HPLC. Amino acid analysis and sequence determination by fast atom bombardment and electrospray tandem mass spectrometry revealed the composition and structures of six 11‐mer peptides of the HM A group and of six 18‐mer peptides of the HM B group. Positions of isomeric amino acids Leu/Ile and Val/Iva (present in some of the peptides) were determined by methanolytic cleavage of the pure peptides, followed by trifluoroacetylation of the dipeptide methyl esters released and assignment of their structures by gas chromatography‐selected ion monitoring mass spectrometry. As examples, two sequences of HM A and HM B are presented (exchange positions in parentheses). – HM A‐1(2): Ac‐Aib<jats:sup>1</jats:sup>(<jats:sc>D</jats:sc>‐Iva<jats:sup>1</jats:sup>)‐Gln‐Val‐Val‐Aib‐Pro‐Leu‐Leu‐Aib‐Pro‐Leuol<jats:sup>11</jats:sup>; HMB‐1(2): Ac‐Aib<jats:sup>1</jats:sup>‐Ser‐Ala‐Leu‐Aib‐Gln‐Aib‐Val‐Aib‐Gly‐Aib‐Pro‐Leu‐Aib‐Aib‐Gln‐Valol<jats:sup>18</jats:sup> (Leuol<jats:sup>18</jats:sup>), (Ac = acetyl, Aib = α‐aminoisobutyric acid, Iva = <jats:sc>D</jats:sc>‐isovaline, Leuol = <jats:sc>L</jats:sc>‐leucinol, Valol = <jats:sc>L</jats:sc>‐valinol).