When growing on N(2), actinomycetes from the genus Frankia form multicellular structures that contain nitrogenase. The structures are referred to as vesicles and are indistinguishable from vesicles formed when Frankia sp. are in root-nodule symbioses. Vesicles isolated from N(2)-grown cells of Frankia sp. strain CpI1 had a significantly higher amount and different composition of fatty acids than did vegetative cells recovered from NH(4) (+)-containing medium. Lipids from vesicles, whole cells grown on N(2), and whole cells grown on NH(4) (+) were fractionated by silicic acid chromatography into neutral lipids, glycolipids, and polar lipids. The fatty acids were transesterified by methanolysis and analyzed by gas chromatography and mass spectrometry. Vesicles had considerably higher amounts of fatty acids in the neutral and glycolipid fractions but lower amounts of polar lipid fatty acids than did vegetative cells. Polar lipids from vesicles had a higher proportion of mono-unsaturated and cyclopropane fatty acids and a lower proportion of isobranched fatty acids than did polar lipids from NH(4) (+)-grown or N(2)-grown cells. The neutral lipid and glycolipid fractions contained several long-chain compounds with molecular ions at m/z 408 and 410. The proportions of these compounds were significantly higher in the lipids from vesicles than from vegetative cells. These results suggest that lipids in vesicles might be involved in the protection of nitrogenase from O(2) and suggest a parallel with the glycolipids involved in protecting nitrogenase from O(2) in the cyanobacterial heterocysts.