Groundwater nitrification is a poorly characterized process affecting the speciation and transport of nitrogen. Cores from two sites in a plume of contamination were examined using culture-based and molecular techniques targeting nitrification processes. The first site, located beneath a sewage effluent infiltration bed, received treated effluent containing O₂(> 300 µM) and NH₄⁺ (51–800 µM). The second site was 2.5 km down-gradient near the leading edge of the ammonium zone within the contaminant plume and featured vertical gradients of O₂, NH₄⁺, and NO₃⁻ (0–300, 0–500, and 100–200 µM with depth, respectively). Ammonia- and nitrite-oxidizers enumerated by the culture-based MPN method were low in abundance at both sites (1.8 to 350 g^− 1 and 33 to 35,000 g^− 1, respectively). Potential nitrifying activity measured in core material in the laboratory was also very low, requiring several weeks for products to accumulate. Molecular analysis of aquifer DNA (nested PCR followed by cloning and 16S rDNA sequencing) detected primarily sequences associated with the Nitrosospira genus throughout the cores at the down-gradient site and a smaller proportion from the Nitrosomonas genus in the deeper anoxic, NH₄⁺ zone at the down-gradient site. Only a single Nitrosospira sequence was detected beneath the infiltration bed. Furthermore, the majority of Nitrosospira-associated sequences represent an unrecognized cluster. We conclude that an uncharacterized group associated with Nitrosospira dominate at the geochemically stable, down-gradient site, but found little evidence for Betaproteobacteria nitrifiers beneath the infiltration beds where geochemical conditions were more variable.