The quantitative contribution of acetic acid to methane production was studied by use of C(14)-labeled acetic acid. Samples of domestic sewage sludge were incubated anaerobically in Warburg vessels. The rate of methane production in the vessels was 0.033 mumoles per ml per min. C(14)-labeled acetic acid was added, and the turnover rate was calculated. The pool size of acetic acid in the sludge was 4.7 mumoles/ml. The turnover rate was 0.0052 min(-1), giving a rate of formation of acetic acid of 0.024 mumoles per ml per min. Under these conditions, acetic acid would account for approximately 73% of the methane produced by the sludge. Acetic acid was found to exist primarily in an extracellular pool. The turnover rate of the extracellular pool was rapid, and it was concluded that most of the acetic acid must be metabolized to methane by a specialized microflora not involved in the formation of acetic acid.