The branch point of the tricarboxylic acid and glyoxylate shunt has been characterized in the intact organism by a multidimensional approach. Theory and methodology have been developed to determine velocities for the net flow of carbon through the major steps in acetate metabolism in Escherichia coli. Rates were assigned based on the 13C NMR spectrum of intracellular glutamate, measured rates of substrate incorporation into end products, the constituent composition of E. coli, and a series of conservation equations which described the system at steady state. The in vivo fluxes through the branch point of the tricarboxylic acid and glyoxylate cycles were compared to rates calculated from the kinetic constants of the branch point enzymes and the intracellular concentrations of their substrates.