Due to reduced evaporation and diffusion of water molecules containing heavier isotopes, leaf water possesses an elevated 18O or 2H steady-state content. This enrichment has been exploited in plant physiology and ecology to assess transpiration and leaf water relations. In contrast to these studies, in this work the 2H content of the medium of hydroponically grown Arabidopsis thaliana was artificially raised, and the kinetics of 2H increase in the aerial parts recorded during a short phase of 6–8 h, until a new equilibrium at a higher level was reached. A basic version of the enrichment models was modified to establish an equation that could be fitted to measured leaf 2H content during uptake kinetics. The fitting parameters allowed estimation of the relative water flux qleaf into the Arabidopsis rosette. This approach is quasi-non-invasive, since plants are not manipulated during the uptake process, and therefore, offers a new tool for integrated analysis of plant water relations. The deuterium tracer method was employed to assess water relocation in Arabidopsis pip2;1 and pip2;2 aquaporin knockout plants. In both cases, qleaf was significantly reduced by about 20%. The organ and cellular expression patterns of both genes imply that changes in root hydraulic conductivity, as previously demonstrated for pip2;2 mutants, and leaf water uptake and distribution contributed in an integrated fashion to this reduced flux in intact plants.