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The sterol analysis of six hydrocarbon seep mussels (mytilid II and mytilid Ib) from the Alaminos Canyon in the Gulf of Mexico is reported. The sterol composition of the mussel-symbiotic bacteria complex reflects a preponderant synthesis of 4 alpha -methyl sterols (seep mytilid II), and a predominant biosynthesis of 4-desmethyl sterols with some amounts of 4 alpha -methyl sterols (seep mytilid Ib). This suggests a methane-based symbiotic relationship between the mussels and methanotrophic bacteria. It also suggests that the biosynthesis of sterols in the mussel-bacteria complex is completed to the level of cholest-5-en-3 beta -ol (mytilid II) or 5 alpha -cholestan-3 beta -ol (mytilid Ib).