ABSTRACI The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C4 plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabo-lites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between I and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function. (23, 27). This derived value for mesophyll-bundle sheath cell resistance has also been applied to different models and given estimated diffusive losses of stored inorganic carbon from bundle sheath cells of the order of 10% of the CO2 originally generated by C4 acid decarboxylation (14, 21). Clearly, a more precise analysis of C4 pathway function would require more reliable direct measurements for the diffusion characteristics of the mesophyll-bundle sheath cell interface. It is difficult to conceive how such measurements might be obtained with intact leaves but, as recently pointed out (3), isolated bundle sheath cells may provide an alternative system. Rapid flux of C4 acids or C3 compounds into isolated bundle sheath cell strands, as judged by resulting rates of metabolism or photosynthesis, have been observed in several separate studies (4, 6, 19, 26). Responses to the addition of various other metabolites and inhibitors have also been observed, including compounds as large as adenylates and NAD (3, 4, 9). During the present study we examined the diffusion of various metabolites into isolated bundle sheath cell strands from C4 species and a procedure was developed for measuring diffusion constants for this process. This diffusive flux apparently occurred via plasmodesmata. The results are considered in relation to the function of C4 photosynthesis. A key feature of C4 plants is the differential partitioning of photosynthetic reactions between mesophyll and bundle sheath cells (21). As a consequence, there is a need for rapid intercellular transport of certain metabolites between these cells. The prime function of C4 photosynthesis is to concentrate CO2 in bundle sheath cells (1, ...