Recent PMR and X-ray studies have shown that a common conformational feature of several cyclic polypeptide antibiotics is the β-turn associated with antiparallel pleated sheet structures. The purpose of this study was to investigate if ilamycin B₁, a cyclic heptapeptide consisting of only L-amino acid residues, can have an antiparallel β-type conformation. Using 300 MHz proton magnetic resonance (PMR) in DMSO-d₆, five amide proton resonances were observed, with two showing low temperature coefficients and slow hydrogen-deuterium exchange, indicative of intramolecular hydrogen bonding. Decoupling experiments assigned resonances to specific residues and revealed a rigid secondary structure. A proposed secondary structure of ilamycin B₁ includes β-turns with hydrogen bonding between the amide proton of 3-nitrotyrosine and the carbonyl of 2-amino-trans-4-hexenoic acid, and between the amide proton of the hexenoic acid and the carbonyl of alanine, explaining the high field shift of a leucyl isopropyl methyl resonance via ring current effects and consistent with slow exchange of amide protons 4 and 5 due to hydrogen bonding.