Bacterial cultures capable of reducing nitrate to nitrite, or of complete denitrification, were established from 5, 10, 15 and 20 cm depths of a freshwater sediment. Taxonomic analysis of the 56 isolates using 16S rRNA gene sequences revealed an unexpected species richness, which included representatives of the γ-Proteobacteria, Bacillus spp., Staphylococcus spp. and members of the Actinobacteria. Gram-positive species tended to predominate in the lower depths of the sediment, where there was evidence of active sulphate respiration. Sequences (from the narG gene) potentially encoding the catalytic subunit of the membrane-associated nitrate reductase were successfully amplified from 46 of the isolates, using a nested PCR with four degenerate primers. NarG sequences clustered into three major groupings that were supported by alternative phylogenetic analyses. The NarG sequences from Gram-positive isolates (according to rRNA gene phylogeny) clustered together within sequences from the low-G+C Gram-positive bacteria. However, this cluster also included two sequences from members of the genus Pseudomonas. Another group contained mostly NarG sequences from the Proteobacteria (according to rRNA gene phylogeny), but also included five sequences from Gram-positive species. The third group of NarG sequences contained three sequences from Gram-positive species. Thus, the NarG-derived phylogeny is not entirely consistent with 16S rRNA-based taxonomy, precluding the use of the narG gene as a taxonomically useful tool for the characterization of nitrate-respiring bacteria. Total DNA was also extracted from the four depth intervals of the sediment sample and used in similar narG amplifications. Most sequences amplified directly from environmental DNA clustered in the Gram-negative group, and none was in the predominantly Gram-positive group. The study also revealed a degree of spatial organization of a nitrate-respiring community in terms of both microbiology and narG sequences.