Biosynthesis of the trisaccharide precursor of fructan occurs through the action of sucrose: sucrose fructosyltransferase (SST). This study defines the optimal conditions for in vitro SST and invertase activities from a crested wheatgrass hybrid developed from a cross between Agropyron cristatum (L.) Gaertn. ×A. desertorum (Fisch. ex Link) Schult. Enzyme extracts were prepared from field-grown plants. The optimum pH for SST and invertase activities were 5.0 and 4.5, respectively. The effective Km for SST (100 mol m−3) is much larger than invertase (15 mol m−3). Sodium pyrophosphate differentially reduced invertase activity without affecting SST activity. Conversely, CaCl2 and CsCl reduced SST activity without altering invertase activity. Optimal in vitro reaction temperatures for SST and invertase were 30 and 50°C, respectively. SST activity was 30% of maximum at temperatures as cool as 5°C. In contrast, invertase activity was only 10% of maximum at 15 °C. The responses of SST and invertase to changes in pH, temperature, and additives provide additional evidence that the two activities are the result of two distinct enzymes. It is hypothesized that cool-season grasses such as crested wheatgrass have the ability to fix carbon photosynthetically at lower temperatures than starch-accumulating species because of their capacity to synthesize fructans.