Perchlorate (004) contamination of ground and surface water has been recently recognized as a widespread environmental problem. Biological methods offer promising perspectives of Perchlorate remediation. Facultative anaerobic bacteria couple the oxidation of organic and inorganic electron-donating substrates to the reduction of Perchlorate as a terminal electron acceptor, converting it completely to the benign end-product, chloride. Insoluble inorganic substrates are of interest for low maintenance bioreactor or permeable reactive barrier systems because they can provide a long-term supply of electron donor without generating organic residuals. The main objective of this research was to investigate the feasibility of utilizing elemental sulfur (SO) as an insoluble electron donor for the biological reduction of perchlorate. A chemolithotrophic enrichment culture derived from aerobic activated sludge was obtained which effectively coupled the oxidation of elemental sulfur to sulfate with the reduction of perchlorate to chloride and gained energy from the process for cell growth. The enrichment culture grew at a rate of 0.41 or 0.81 1/d in the absence and presence of added organic carbon for cell growth, respectively. The enrichment culture was also shown to carry out sulfur disproportionation to a limited extent as evidenced by the formation of sulfide and sulfate in the absence of added electron acceptor. When nitrate and Perchlorate were added together, the two electron acceptors were removed simultaneously after an initial partial decrease in the nitrate concentration.