Anaerobic toxicity assays were performed with 150 ml serum bottles using acetate-utilizing methanogenic enrichment cultures spiked with cobalt at 35–1500 mg-l−1. Free cobalt concentrations in the bottles were measured by a combination of dialysis and ion exchange at the end of the experiments. Free cobalt and total soluble cobalt of 70 and 280 mg-l−1, respectively, caused complete inhibition of methanogenesis. Free cobalt/VSS and total soluble cobalt/VSS ratios of and exceeding 0.05 and 0.16, respectively, caused complete failure of methanogenesis.