Background and Aims Regulation of water channel aquaporins (AQPs) provides another mechanism by which abscisic acid (ABA) may influence water flow through plants. To the best of our knowledge, no studies have addressed the changes in ABA levels, the abundance of AQPs and root cell hydraulic conductivity (Lp(Cell)) in the same tissues. Thus, we followed the mechanisms by which ABA affects root hydraulics in an ABA-deficient barley mutant Az34 and its parental line 'Steptoe'. We compared the abundance of AQPs and ABA in cells to determine spatial correlations between AQP abundance and local ABA concentrations in different root tissues. In addition, abundance of AQPs and ABA in cortex cells was related to Lp(Cell). Methods Root hydraulic conductivity (Lp(Root)) was measured by means of root exudation analyses and Lp(Cell) using a cell pressure probe. The abundance of ABA and AQPs in root tissues was assessed through immunohistochemical analyses. Isoform-specific antibodies raised against HvPIP2; 1, HvPIP2; 2 and HvPIP2; 5 were used. Key Results Immunolocalization revealed lower ABA levels in root tissues of Az34 compared with ` Steptoe'. Root hydraulic conductivity (Lp(Root)) was lower in Az34, yet the abundance of HvPIPs in root tissues was similar in the two genotypes. Root hair formation occurred closer to the tip, while the length of the root hair zone was shorter in Az34 than in ` Steptoe'. Application of external ABA to the root medium of Az34 and ` Steptoe' increased the immunostaining of root cells for ABA and for HvPIP2; 1 and HvPIP2; 2 especially in root epidermal cells and the cortical cell layer located beneath, parallel to an increase in Lp(Root) and Lp(Cell). Treatment of roots with Fenton reagent, which inhibits AQP activity, prevented the ABA-induced increase in root hydraulic conductivity. Conclusion Shortly after (<2 h) ABA application to the roots of ABA-deficient barley, increased tissue ABA concentrations and AQP abundance (especially the plasma-membrane localized isoforms HvPIP2; 1 and HvPIP2; 2) were spatially correlated in root epidermal cells and the cortical cell layer located beneath, in conjunction with increased Lp(Cell) of the cortical cells. In contrast, long-term ABA deficiency throughout seedling development affects root hydraulics through other mechanisms, in particular the developmental timing of the formation of root hairs closer to the root tip and the length of the root hair zone.