A toluene degrading denitrifier bacterium, A. tolulyticus Tol-4, was I of 8 similar strains isolated from 3 petroleum-contaminated aquifer sediments. When the strain is grown anaerobically on toluene, 68% of the carbon from toluene is found as carbondioxide and 30% is found as biomass. Because this organism is an obligate anaerobe, it must have a novel pathway for biodegradation of toluene. Benzoic acid appears to be an intermediate in the pathway, because fluoro-benzoate accumulated when fluorotoluene was added to the growth medium. In addition, [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 -2%) and benzy1succinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzyl alcohol, benzaldehyde, benzoic acid, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl-CoA attack in strain Tol-4. We propose that a pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzy1succinic acid to form E-phenylitaconic acid. We propose to prepare some of thes intermediates labeled with carbon- 13 and follow their metabolism by carbon- 13 NMR spectroscopy.