To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).