Hexavalent Chromium is a widespread contaminant found in soil, sediment, and groundwater. In order to stimulate microbially mediated reduction of Cr(VI), a poly-lactate compound HRC was injected into the chromium-contaminated aquifers at site 100H at Hanford, WA. Based on the results of the bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products, we investigated the diversity of the dominant anaerobic culturable microbial populations present at this site and their role in Cr(VI) reduction. Also, functional gene array (GeoChip) analysis of DNA extracted from monitoring well at the site indicated that genes involved in nitrate reduction, sulfate reduction, iron reduction, methanogenesis, as well as many chromium tolerance/reduction genes were highly abundant relative to the injection well. In addition, positive enrichments set up at 30oC using defined anaerobic media resulted in the isolation of an iron-reducing isolate strain HAF, a sulfate-reducing isolate strain HBLS and a nitrate-reducing isolate, strain HLN among several others. Preliminary 16S rDNA sequence analysis identified strain HAF as Geobacter metallireducens, strain HLN as Pseudomonas stutzeri and strain HBLS as Desulfovibrio species. Strain HAF utilized propionate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Growth was optimal at 37oC and pH of 6.5. Strain HLN utilized acetate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Optimal growth was observed at 37oC, at a pH of 7.5. Anaerobic washed cell suspension of strain HLN reduced almost 95 muM Cr(VI) within 4 h relative to controls. Further, with 100 muM Cr(VI) as sole electron acceptor, cells of strain HLN grew to cell numbers of 4.05 x 107/ml over a period of 24 h after an initial lag, demonstrating direct enzymatic Cr(VI) reduction by this species. These results demonstrate that Cr(VI) immobilization at the Hanford 100H site could be mediated by direct microbial metabolism apart from indirect chemical reduction of Cr(VI) by end products of microbial activity.