Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene. A possible approach to clean-up VC-contaminated sites is with microorganisms that grow on VC and/or ethene as a carbon and energy source. However, little is known about the biochemical pathways involved in VCassimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract and identify proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. These techniques were extended to 14 ethene-enrichment cultures derived from VC-contaminated groundwater to detect the presence and diversity of VC-and/or ethene-assimilating bacteria with previously identified protein biomarkers. VC-assimilating organisms can evolve under laboratory conditions from bacteria that grow on ethene but very little is known about the molecular changes involved. Tandem mass spectrometry and spectral counting were applied to three variants of ethene-grown Mycobacterium strain JS623 (a wild type and two VC-adapted variants) resulting in the identification of 174 differentially expressed proteins. The results of this v ABSTRACT Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene. A possible approach to clean-up VC-contaminated sites is with microorganisms that grow on VC and/or ethene as a carbon and energy source. However, little is known about the biochemical pathways involved in VCassimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract a...