The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.Compartmentation is one of the distinguishing characteristics of plant metabolism (ap Rees, 1987). A true understanding of the nature and regulation of plant metabolic networks can only be achieved when the metabolic interactions between subcellular compartments have been charted and subjected to analysis through experimental procedures. Because of the profound difficulties associated with measuring enzymes, metabolites, and fluxes in specific subcellular compartments, our understanding of plant metabolism has lagged far behind that of animal and microbial systems.Although methods have been developed for the assay of subcellular metabolite levels in leaf tissue (Stitt et al., 1989), and the interactions between plastidial and cytosolic metabolism during photosynthesis have been partially characterized (Stitt, 1997), little is known about the metabolic networks in heterotrophic cells. There are two main reasons for this. First, there is a lack of suitable methods for organelle isolation, which is a particularly difficult problem in heterotrophic cells because these often contain large starch grains that cause extra damage to the organelles during fractionation. Second, although leaf metabolism is highly conserved between different species (Heineke et al., 1997), heterotrophic tissues usually form differentiated organs with specific functions and therefore studies ca...