The interaction between microbially-mediated nitrogen cycling and catchment hydrology affects the amount and isotopic composition of nitrate exported from catchments in drainage waters. Dominant microbial and hydrological influences were investigated using delta N-15(NO3) and delta O-18(NO3) of nitrate from the Wensum catchment in East Anglia, eastern England; a 570 km(2) lowland agricultural catchment. Samples were collected from catchment waters, precipitation, dry deposition, agricultural fertiliser and sewage effluent. Catchment water nitrate concentration and isotopic composition can be explained by microbially-mediated cycling of nitrogen inputs through nitrification to denitrification, resulting in a reduced nitrate load exported from the Wensum catchment. Seasonal, transient and through-year constant isotopic signals from nitrogen cycling processes reflect the influence of dynamic and stable hydrological factors. A three-member mass-balance mixing model demonstrates an increasing influence from Chalk groundwater downstream in the Wensum headwaters, and the displacement of shallow groundwater into the river by runoff explains the isotopic and hydrochemical stability seen in the river Wensum under varying flow conditions. Together this demonstrates a powerful application of a dual isotope and hydrological approach in the understanding of an agricultural catchment's response to nitrogen loading.