Hydrogen (H2) is produced and utilized by a large number of microorganisms in anaerobic sediments, and acts as a metabolic link between microbes with very diverse substrate requirements. In environments with low organic carbon input H2 is often the major reductant available to microbes. The importance of H2 suggests that enzymatic (catalytic) activity involved in its metabolism is a good index of overall microbial community metabolism in anaerobic environments. To test this hypothesis, we have developed a radiotracer assay for hydrogenase, an enzyme that all H2-producing and H2-consuming microbes possess. The assay is suitable for water as well as for sediment samples. Our method relies on the ability of hydrogenase to catalyze isotopic exchange between dissolved tritiated H2 and water. The rate of accumulation of tritium in the water is proportional to the amount of enzyme present. A pilot experiment conducted on Leg 201 of the Ocean Drilling Program suggested that hydrogenase activity varied consistently with the rate of sulfate reduction estimated by modeling of pore water sulfate concentration profiles in the sediment column. To simulate environmental samples with very low hydrogenase activity, diluted cultures or coastal sediments were used as test material. Present work is focused on 1) processing samples from recent IODP cruises to the Porcupine Basin and the Gulf of Mexico; 2) producing a standard hydrogenase preparation for use in assay development and quality control; 3) investigating the stability of hydrogenase activity in frozen samples; and 4) increasing the sensitivity of the procedure.