Several enrichment cultures were investigated in our laboratory for the bioremediation potential of tetrachloroethene (PCE). The underlying process, organohalide respiration (OHR), is based on bacterial anaerobic respiration in which the chlorinated compounds are used as electron acceptors. The key enzyme in OHR is the reductive dehalogenase (RdhA). SL2-PCEb, an enrichment culture stepwise dechlorinating PCE to cis-dichloroethene (cis-DCE), was shown to be dominated by Sulfurospirillum spp. By applying different frequencies of culture transfer, two additional subcultures were obtained showing different degradation properties. The aim of this work was to obtain a better understanding of the functional diversity of reductive dehalogenases (rdhA genes) in the SL2 consortia and their dynamic during PCE dechlorination. An rdhA-specific T-RFLP method was developed here to amplify all rdhA gene types present in Sulfurospirillum, and to distinguish them across the dechlorination patterns. The analysis of subcultures SL2-PCEc and SL2-TCE revealed that the former only dechlorinated PCE to trichloroethene (TCE), while the latter had kept the potential to dechlorinate PCE to cis-DCE. Three rdhA genes were identified in SL2 subcultures, two of them similar to Type-1 rdhA genes (pceA of S. multivorans and S. halorespirans), and a third one to Type-2. The T-RFLP analysis of the original culture (SL2-PCEb) showed a clear interplay of both Type-1 rdhA genes, highlighting a distinct role for each of them in the stepwise dechlorination of PCE. Biochemical characterization of crude extracts from the subcultures unambiguously confirmed these observations. Moreover, a new RdhA enzyme was discovered with a limited substrate range.