The uptake of C4 dicarboxylates by cells from exponential cultures of Rhodopseudomonas spheroides followed saturation kinetics at concentrations below 100 muM with Km values for succinate, malate, and fumarate of 2.7, 2.3, and 0.8, respectively. Corresponding Vmax values of 50, 52, and 67.5 nmol/min per mg of protein at 20 C were obtained. Each of these compounds interfered competitively with uptake of the others, and a common transport system appears to be involved. Fructose-grown cells took up C4 dicarboxylates only at very low rates, and pyruvate-grown cells took up C4 dicarboxylates at one-third the rates found with succinate-grown cultures. Malonate and maleate inhibited uptake less severely, and aspartate and alpha-ketoglutarate had no effect at 100-fold excess. Divalent metals stimulated uptake. Light or respiration was required for uptake, and entered materials were rapidly converted to other metabolities, notably amino acids. Pyruvate entry appeared to be mediated by several systems, of which only one could be resolved kinetically. This system had a Km of 13 muM and Vmax of 5.6 nmol/min per mg of protein at 20 C. A number of related mono- and dicarboxylates interfered with pyruvate uptake. The pyruvate uptake system was distinguishable from the C4 dicarboxylate system by the absence of divalent cation stimulation and by substrate and inhibitor specificity.