Studies of water stress commonly examine either gas exchange or leaf metabolites, and many fail to quantify the concentration of CO2 in the chloroplasts (Cc). We redress these limitations by quantifying Cc from discrimination against 13CO2 and using gas chromatography–mass spectrometry (GC–MS) for leaf metabolite profiling. Five Eucalyptus and two Acacia species from semi-arid to mesic habitats were subjected to a 2 month water stress treatment (Ψpre-dawn = −1.7 to −2.3 MPa). Carbohydrates dominated the leaf metabolite profiles of species from dry areas, whereas organic acids dominated the metabolite profiles of species from wet areas. Water stress caused large decreases in photosynthesis and Cc, increases in 17–33 metabolites and decreases in 0–9 metabolites. In most species, fructose, glucose and sucrose made major contributions to osmotic adjustment. In Acacia, significant osmotic adjustment was also caused by increases in pinitol, pipecolic acid and trans-4-hydroxypipecolic acid. There were also increases in low-abundance metabolites (e.g. proline and erythritol), and metabolites that are indicative of stress-induced changes in metabolism [e.g. γ-aminobutyric acid (GABA) shunt, photorespiration, phenylpropanoid pathway]. The response of gas exchange to water stress and rewatering is rather consistent among species originating from mesic to semi-arid habitats, and the general response of metabolites to water stress is rather similar, although the specific metabolites involved may vary.