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Abstract Plant hydraulics is key for plant survival and growth because it is linked to gas exchange and drought resistance. Although the environment influences plant hydraulics, there is no clear consensus on the effect of nitrogen (N) supply, which may be, in part, due to different hydraulic conductance normalization criteria and studied species. The objective of this study was to compare the variation of root hydraulic properties using several normalization criteria in four pine species in response to three contrasting N fertilization regimes. We studied four closely related, yet ecologically distinct species: Pinus nigra J.F. Arnold, Pinus pinaster Ait., Pinus pinea L. and Pinus halepensis Mill. Root hydraulic conductance (Kh) was measured with a high-pressure flow meter, and values were normalized by total leaf area (leaf specific conductance, Kl), xylem cross-section area (xylem specific conductance, Ks), total root area (root specific conductance, Kr) and the area of fine roots (fine root specific conductance, Kfr). Controlling for organ size differences allowed comparison of the hydraulic efficiency of roots to supply or absorb water among fertilization treatments and species. The effect of N on the root hydraulic efficiency depended on the normalization criteria. Increasing N availability reduced Kl and Ks, but increased Kh, Kr and especially Kfr. The positive effect of N on Kr and Kfr was positively related to seedling relative growth rate and was also consistent with published results at the interspecific level, whereby plant hydraulics is positively linked to photosynthesis and transpiration rate and fast growth. In contrast, normalization by leaf area and xylem cross-sectional area (Kl and Ks) reflected opposite responses to Kr and Kfr. This indicates that the normalization criteria determine the interpretation of the effect of N on plant hydraulics, which can limit species and treatment comparisons.