The vadose zone of the Chalk aquifer from two sites of different land use was found to contain large numbers of nitrate‐reducing and ammonium‐oxidizing bacteria. Relationships between the type of bacteria and nitrogen compounds produced showed that denitrification was occurring beneath the permanent grassland site, whereas the vadose zone beneath the fertilized arable site was essentially aerobic and little attenuation of nitrate by denitrification was occurring. The results show that nitrate is certain to be involved in biological reactions in the vadose zone of the Chalk, although quantification of the rate and the extent of these reactions is still not possible.