To study the interdependence of sucrose accumulation and its hydrolyzing enzyme, soluble acid invertase (AI; EC 220.127.116.11), in fructan-accumulating temperate grasses and cereals, experiments were performed in which sucrose synthesis was abolished in leaves of Lolium temulentum by four independent inhibitory factors, each having a distinct mechanism of action. Trials in the light with mannose or vanadate and in the dark with anoxia or cyanide showed that previously accumulated sucrose was stable in the tissue over a 5- to 6-h period. Conversely, putatively vacuolar AI activity in tissue homogenates was sufficient to completely convert endogenous sucrose to monosaccharide within the same period. Continuous invertase-mediated breakdown of sucrose was thus not a feature of this tissue. It is concluded that AI and sucrose were not in metabolic contact in vivo, implying differential compartmentation. In darkness, in uninhibited leaves, sucrose concentrations fell linearly with respect to time at a rate of –0.6 mg g–1 FW h–1, over a 5- to 6-h period. This value is equivalent to rates of dark respiration measured by gas exchange. Dark-utilisation of sucrose was not accompanied by monosaccharide accumulation in the tissue. The rate of sucrose loss was 3-fold lower than rates of extractable AI activity. Hence, if AI was involved in dark-utilisation, then this implies at least a partial differential localisation of enzyme and substrate. However, the dark-consumption of sucrose was completely abolished by anoxia and by cyanide. It follows that dark-mobilisation (unlike invertase hydrolysis per se) was respiration-dependent and did not result from a simple co-localisation of sucrose and invertase. Taken together, the results show that sucrose and invertase do not share the same metabolic compartment in grass leaves. It is possible that invertase has no role in the mobilisation of stored sucrose in leaves of the fructan-accumulating grasses.