Compartmental analyses of intact roots of barley (Hordeum vulgare L. cv. Klondike) plants, grown with different levels of NO3− (up to 1·0 mol m−3) in the external media, were undertaken using 13NO3−. Two additional treatments, namely sodium dodecyl sulphate (SDS) or brief exposure to high temperature, designed to investigate the identity of the three NO3− compartments revealed by compartmental analyses, provided support for the identification of the latter as corresponding to superficial solution, apoplasm, and cytoplasm. Half-lives for exchange of these compartments, 3 s, 30 s, and 7 mm, were unaffected by the level of NO3− provided during growth. Independent estimates of 13NO3− fluxes obtained by direct methods agreed well with values of fluxes calculated from the compartmental analyses. Cytoplasmic [NO3−], estimated from the compartmental analyses, were in the range from 1–37 mol m−3, and increased with increasing [NO3−] of the medium. Such values for cytoplasmic [NO3−] are inconsistent with an earlier proposal (Siddiqi, Glass, Ruth, and Rufty, 1990; Glass, Siddiqi, Ruth, and Rufty, 1990) of passive NO3− uptake in the concentration range above 10 mol m−3. A model, based upon localized distribution of nitrate reductase activity in epidermal cells, is proposed in which the proposed passive low affinity NO uptake at high external [NO3−] is restricted to epidermal cells. During loading periods with 13NO3−, significant amounts of 13N were translocated to the shoot. Two pools of 13N, one being the root symplasm, appear to participate in the transfer of labelled N to the shoot.