A region on the Methylobacterium extorquens AM1 chromosome previously shown to complement a chemically induced mutant (PCT48) unable to convert acetyl-CoA into glyoxylate was characterized in detail in order to identify the gene(s) involved in the unknown pathway for acetyl-CoA oxidation. Six complete and two partial ORFs were identified by sequencing. Sequence comparisons suggested these might code for, respectively, a dehydrogenase of unknown specificity, a polypeptide of at least 15 kDa with unknown function, a coenzyme-B12-linked mutase, a catalase, an alcohol dehydrogenase (ADH) of unknown function, a polypeptide of 28 kDa, a ketol-acid reductoisomerase and a propionyl-CoA carboxylase (PCC). Insertion mutations were introduced into each ORF in order to determine their involvement in C1 and C2 metabolism. Mutations in three genes, encoding the mutase, ADH and PCC, resulted in a phenotype characteristic of mutants unable to oxidize acetyl-CoA, i.e. they were C1-and C2-negative and their growth on these compounds was restored by the addition of glycolate or glyoxylate. Mutants in the genes thought to encode catalase and PCC were found to be deficient in the corresponding enzyme activity, confirming the identity of these genes, while physiological substrates for the mutase and ADH remain unidentified. This work, in which three new genes necessary for conversion of acetyl-CoA into glyoxylate were identified, is an intermediary step on the way to the solution of the unknown pathway for acetyl-CoA oxidation in isocitrate-lyase-negative methylotrophs.