The structure of an ornithine-containing lipid from Cytophaga johnsonae Stanier strain C21 was determined by ¹H-NMR analysis of its major molecular species (compound 1) consisting of iso-3-hydroxy heptadecanoic (iso-3-OH-17:0) and iso-pentadecanoic acids (iso-15:0). The NOESY spectrum showed the cross peaks between α-NH of ornithine and two protons of the α-methylene moiety of iso-3-OH-17:0. The α-amino group of the ornithine did not form an amide linkage with any fatty acid. Thus, the structure of the ornithine-containing lipid was iso-3-hydroxy heptadecanoic acid, α-amide linked to ornithine and esterified to iso-15:0.