ABSTRACT This study examined osmotic adjustment (OA) and solutes accumulated in leaf segments of wheat cultivar Hartog exposed to polyethylene glycol (PEG)-induced water deficit (WD) in laboratory experiments. Additional 10 mM K+ or Na+ added to basal solution with PEG −0.5 MPa enhanced leaf OA by up to 100%. Omission of nitrogen from the basal solution suppressed the expression of OA in leaf segments, but the addition of 10 mM NO3 – increased leaf OA by up to 81%. K+ and glycinebetaine were not accumulated in leaf segments exposed to WD under PEG −0.5 MPa during 48 h, but Na+, Cl−, proline and total soluble sugars accounted for up to 21, 20, 7 and 40% of OA, respectively. Total soluble sugars were the largest contributor to OA and may also contribute to membrane stability. Further data are needed on accumulation of leaf NO3 − and glycinebetaine in response to increasing nitrogen supply, and on which other inorganic and organic solutes may also contribute to OA under PEG-induced WD. Laboratory experiments with leaf segments exposed to WD under −0.5 MPa PEG, and with 10 mM K+, Na+ or NO3 − added to the basal solution, could provide an effective and rapid pre-screen of diverse germplasm sources for OA expression. Selected genotypes should then be validated by exposure to soil WD for agronomic evaluation, and for OA expression under field WD.