Nitrate contamination of groundwater aquifers from agricultural fertilization is of growing concern. It is generally assumed that little biological activity occurs in the vadose zone and that NO−3moves through this zone unimpeded. Various N fractions and the microbial activity in the surface and vadose zone sediments from three agricultural soils in eastern Oregon were examined. Nitrate levels in the vadose zone were as high as 60 mg N kg−1soil, and fractionation showed that approximately 30% of total N was in the form of acid-insoluble N, which could be a sink for NO−3that becomes immobilized. The vadose zone also contained high concentrations of amino acid-N and amino sugar-N, suggesting that a significant amount of subsoil N is of biological origin. Significant levels of microbial biomass C and CO2respiration were found in sediments derived from the vadose zone. A 14-day lab incubation study showed that vadose zone sediments could mineralize organic N without additional C and that NO−3may be subject to biological immobilization when a high rate of glucose C (3000 mg kg−1) is present.