Understanding where nitrate is mobilized from and under what conditions is required to reduce nitrate loss and protect water quality. Low frequency sampling may inadequately capture hydrological and bio-geochemical processes that will influence nitrate behavior. We used high-frequency isotope sampling and in-situ nitrate sensing to explore nitrate export and transformation in a karst critical zone. Nitrate was mobilised during light rainfall, and transferred from soil layers to the karst matrix, where some nitrate was retained and denitrified. Nitrate isotopic composition changed rapidly during the rising limb of events and slowly during the falling limb. The main nitrate source was synthetic fertiliser (up to 80% during event flow), transported by conduit flow following high rainfall events, and this contribution increased significantly as discharge increased. Soil organic nitrogen contribution remained constant indicating at baseflow this is the primary source. Isotope source appointment of nitrate export revealed that synthetic fertilizer accounted for more than half of the total nitrate export, which is double that of the secondary source (soil organic nitrogen), providing valuable information to inform catchment management to reduce nitrate losses and fluvial loading. Careful land management and fertilizer use are necessary to avoid nitrate pollution in the karst agroecosystem, for example by timing fertilizer applications to allow for plant uptake of nitrate before rainfall can flush it from the soils into the karst and ultimately into catchment drainage. (c) 2020 The Authors. Published by Elsevier Ltd.