Studies with mutants of Salmonella typhimurium and Escherichia coli which fail to grow on glucose-salts media unless supplemented with tricarboxylic acid cycle intermediates or their precursors have shown that the carboxylation of phosphoenolpyruvate (PEP) is necessarily involved in maintaining the tricarboxylic acid cycle by replenishing it with C4-compounds as intermediates are removed during biosynthesis. Although phosphoenolpyruvate carboxylase activity in crude bacterial extracts was initially found insufficient for its postulated role, higher isotope incorporation from labelled bicarbonate was observed when extracts were incubated with acetate, ATP, and CoASH. Experiments with E. coli mutant AB 1623 (unable to catalyse citrate formation from CoASAc and oxaloacetate) showed that catalytic quantities of CoASAc stimulated the rate of oxaloacetate formation from PEP and bicarbonate by over fifteen-fold. These results explain why the enzyme was previously detected in low quantities and demonstrate a "fine control" mechanism for this anapleratic sequence: CoASAc, a product of PEP catabolism, stimulates PEP carboxylation, balancing PEP transformation to CoASAc and oxaloacetate to ensure complete CoASAc combustion and meet the organism's metabolic needs. This control mechanism is similar to that in mammalian systems but differs in using PEP (rather than pyruvate) and not being inhibited by avidin.