The genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples were determined in order to show in parallel the existing and active members of Desulfovibrio populations. DNA and total RNA were extracted from different anaerobic bioreactor samples; RNA was transcribed into cDNA. Subsequently, PCR was performed to amplify a ca.-440-bp fragment of the [NiFe] hydrogenase large-subunit gene and its mRNA. Denaturing gradient gel electrophoresis analysis was used to separate the PCR products according to their sequence and thereby to visualize the individual community members. Desulfovibrio strains corresponding to amplified [NiFe] hydrogenase transcripts were regarded as metabolically active, because in pure cultures transcripts were detectable in exponentially growing cells but not in cultures in the stationary phase. DNA sequencing and comparative sequence analysis were used to identify the detected organisms on the basis of their [NiFe] hydrogenase sequences. The genes of characterized Desulfovibrio spp. showed a considerable extent of divergence (ca. 30%), whereas sequences obtained from bacterial populations of the bioreactors showed a low level of variation and indicated the coexistence of closely related strains probably belonging to the species Desulfovibrio sulfodismutans. Under methanogenic conditions, all detected populations were active; under denitrifying conditions, no [NiFe] hydrogenase mRNA was visible. Changes in activity and composition of Desulfovibrio populations caused by changes in the environmental conditions could be monitored by using the approach described in this study.