The biochemical bases of water-stress tolerance in a pedigree consisting of black cottonwood (PopulustrichocarpaTorr. & Gray female) and eastern cottonwood (PopulusdeltoidesBartr. male) parental clones and four hybrid progeny were investigated. Trees were grown outdoors in pots; well-watered trees (soil water potential greater than −0.03 MPa) were kept moist in trays, and stressed trees (soil water potential less than −2.0 MPa) were subjected to repeated cyclical stress of 1 or 2 days duration over the 14-week study. Analysis of the major metabolites and ions in fully expanded leaves demonstrated that the greatest degree of osmotic adjustment was displayed by male hybrid 242, theP.deltoidesmale parent, and male hybrid 239 to a lesser extent. Osmotic adjustment in leaves of both hybrid 242 and theP.deltoidesmale parent was primarily constituted by malic acid, K, sucrose, and glucose, with the same metabolites also increasing in fine roots of hybrid 242, the only clone to display osmotic adjustment in roots. Female clone 240 andP.deltoidesdisplayed organic solute-based adjustments to water stress that were offset by declines in inorganic ions, particularly Na and Mg. Given that theP.trichocarpafemale parent did not display osmotic adjustment in either tissue, the hybrids' capacity for adjustment was likely conferred by theP.deltoidesmale parent.