The hopanoid content of the two methanotrophic bacteria Methylocaldum szegediense and Methylocaldum tepidum was investigated. 35-Aminobacteriohopane-30R,31R,32R,33S,34S-pentol and its 3β-methyl homologue were present in both strains. In M. tepidum, they were accompanied by 35-aminobacteriohopane-31R,32R,33S,34S-tetrol and its 3β-methyl homologue. The side chain structure was identical to those previously reported from two other obligate methanotrophs, Methylococcus capsulatus and Methylomonas methanica. The two Methylocaldum species shared with the Methylococcus species the presence of 3β-methylhopanoid as well as of a hopanoid releasing adiantol upon H5IO6/NaBH4 treatment. A rare feature was in addition found in M. szegediense. The saturated hopanoids were accompanied by an unsaturated aminobacteriohopanepentol with a Δ11 double bond. Comparison of the hopanoid fingerprints was in accordance with the close phylogenetic relationship of Methylococcus and Methylocaldum. The major difference was the absence of sterols in Methylocaldum which were always detected in the Methylococcus species.