The occurrence of positional isomers of minor C(24) unsaturated fatty acids in female gonad lipids of the limpet Cellana grata was clarified by gas chromatography-mass spectrometry of the combination of their 4,4-dimethyloxazoline and picolinyl ester derivatives. In this study, in addition to 5,9-24:2, 9,15-24:2, 5,9,15-24:3, and 5,9,17-24:3, previously identified, 24:4n-6, 24:5n-3, and four novel nonmethylene-interrupted fatty acids, 9,17-24:2, 9,15,18-24:3, 5,9,15,18-24:4, and 5,9,15,18,21-24:5, were newly recognized. All C(24) unsaturated fatty acids detected were present only in triacylglycerols.