The upper pathway of anaerobic degradation of 2-methylnaphthalene was studied with a sulphate-reducing enrichment culture, which is able to grow with naphthalene or 2-methylnaphthalene as sole carbon source and electron donor. Anaerobic degradation of 2-methylnaphthalene is initiated by an addition of fumarate to the methyl-group producing the first intermediate, naphthyl-2-methyl-succinate. In a subsequent beta-oxidation of the original methyl atom, the central metabolite 2-naphthoic acid is generated. In the following pathway, the aromatic ring system is reduced, cleaved, and finally oxidised to CO(2). Here, we present two new enzymatic reactions of the 2-methylnaphthalene degradation pathway that were measured in crude cell extracts. All metabolites were identified with HPLC by co-elution with synthesised reference substances. The first enzyme, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase, catalyses the activation of naphthyl-2-methyl-succinic acid to the corresponding CoA ester. The average specific activity of this enzyme was 19.6 nmol x min(-1) x mg of protein(-1). The CoA-transfer was not inhibited by sodium borohydride and only partially by hydroxylamine, indicating that this enzyme belongs to the family III of CoA-transferases like the corresponding enzyme in the anaerobic toluene degradation pathway. The product of this CoA-transfer reaction, naphthyl-2-methyl-succinyl-CoA is then oxidised in a reaction to naphthyl-2-methylene-succinyl-CoA by the enzyme naphthyl-2-methyl-succinyl-CoA dehydrogenase. The specific activity of this enzyme was 0.115 nmol x min(-1) x mg of protein(-1). The enzymatic activity could only be detected using phenazine methosulphate as electron acceptor. No activity was observed with natural electron acceptors such as nicotinamide adenine dinucleotide or flavin adenine dinucleotide. The two novel reactions presented here demonstrate that the original methyl-group of 2-methylnaphthalene is oxidised to the carboxyl group of 2-naphthoic acid in the upper part of the anaerobic degradation pathway.