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Anoxic samples (sediment and groundwater) from 13 chemically diverse field sites were assayed for their ability to consume acetylene (C2H2). Over incubation periods ranging from ∼ 10 to 80 days, selected samples from 7 of the 13 tested sites displayed significant C2H2removal. No significant formation of ethylene was noted in these incubations; therefore, C2H2consumption could be attributed to acetylene hydratase (AH) rather than nitrogenase activity. This putative AH (PAH) activity was observed in only 21% of the total of assayed samples, while amplification of AH genes from extracted DNA using degenerate primers derived fromPelobacter acetylenicusoccurred in even fewer (9.8%) samples. Acetylene-fermenting bacteria were isolated as a pure culture from the sediments of a tidal mudflat in San Francisco Bay (SFB93) and as an enrichment culture from freshwater Searsville Lake (SV7). Comparison of 16S rDNA clone libraries revealed that SFB93 was closely related toP. carbolinicus, while SV7 consisted of several unrelated bacteria. AH gene was amplified from SFB93 but not SV7. The inability of the primers to generate amplicons in the SV7 enrichment, as well as from several of the environmental samples that displayed PAH activity, implied that either the primers were too highly constrained in their specificity or that there was a different type of AH gene in these environmental samples than occurs inP. acetylenicus. The significance of this work with regard to the search for life in the outer Solar System, where C2H2is abundant, is discussed. [ABSTRACT FROM PUBLISHER]