The total fatty acid composition of various strains of Hyphomicrobium, Pedomicrobium, and Rhodomicrobium spp. was determined by gas chromatography. In addition, the fatty acid pattern of a new hyphal budding bacterium, strain F-1, was compared with the other patterns obtained. Octadecenoic acid was the main component in most strains, comprising up to 75% of the total fatty acids. Lactobacillic acid and 3-methoxy-tetradecanoic acid were present in varying amounts in the lipids of all organisms except for the new isolate, F-1. This latter strain contained, however, large amounts of iso-heptadecanoic and iso-heptadecenoic acids, not present in the other budding bacteria studied. This composition was consistently found under various culture conditions. The data indicate that, except for the new bacterium F-1, the hyphal budding bacteria studied here are closely related. The total fatty acid composition is thought to be a useful taxonomic criterion for differentiation of these bacteria. © 1979 Springer-Verlag.