Degenerate and specific PCR primers were designed for the detection of chloroethene reductive dehalogenases (CE-RDase), the key enzymes of chloroethene dehalorespiration, based on sequence information of three CE-RDases and three chlorophenol (CP) RDases. For the design of the degenerate primers, seven conserved amino-acid blocks identified with different bioinformatic tools were used. For one block degenerate, primers containing a 5'-consensus clamp region specific for CE-RDases and a 3'-end degenerate core region specific for RDases in general were designed using the Consensus-Degenerate Hybrid Oligonucleotide Primer (CDHOP) design method. Applying the degenerate primers to genomic DNA of Sulfurospirillum multivorans strain K, Dehalobacter restrictus strain PER-K23, and Desulfitobacterium sp. strain PCE1 led to the isolation of the known CE-RDase genes and three new genes encoding putative reductive dehalogenases that cluster with CE-RDases and not with CP-RDases. In addition, primers designed to be specific for the three known CE-RDase genes, namely pceA of S. multivorans, pceA of D. restrictus, and tceA of Dehalococcoides ethenogenes were successfully tested on genomic DNA of different chloroethene-dehalorespiring bacteria. Nested PCR using degenerate primers followed by a PCR with specific primers allowed a sensitive detection of only 102 copies per reaction. (C) 2003 Elsevier B.V. All rights reserved.