Acetate is the most important intermediate in anaerobic degradation of organic matter. The carbon isotope effects associated with the oxidation of acetate (epsilon(ac)) were examined for four acetotrophic sulfur reducers, Desulfuromonas acetoxidans, Desulfuromonas thiophila, Desulfurella acetivorans, and Hippea maritima. During the consumption of acetate and sulfur, acetate was enriched in (13)C by 11.5 and 11.2 per thousand in Desulfuromonas acetoxidans and Desulfuromonas thiophila, respectively. By contrast, isotope fractionation in D. acetivorans and H. maritima resulted in isotope enrichment factors of epsilon(ac)=-6.3 per thousand and -8.4 per thousand, respectively. These sulfur-reducing bacteria all metabolize acetate via the tricarboxylic acid cycle, but have different mechanisms for the initial activation of acetate. In Desulfuromonas acetoxidans, acetyl-CoA is formed by succinyl-CoA : acetate-CoA-transferase, and in D. acetivorans by acetate kinase and phosphate acetyltransferase. Hence, values of epsilon(ac) seem to be characteristic for the type of activation of acetate to acetyl-CoA in acetotrophic sulfur reducers. Summarizing epsilon(ac)-values in anaerobic acetotrophic microorganisms, it appears that isotope fractionation depends on the mechanism of acetate activation to acetyl-CoA, on the key enzyme of the acetate dissimilation pathway, and on the bioavailability of acetate, which all have to be considered when using delta(13)C of acetate in environmental samples for diagnosis of the involved microbial populations.