Citrate synthase (EC 4.1.3.7) was varied from 10% to 5000% the level found in wild-type Escherichia coli by means of recombinant DNA techniques. When acetate was the sole carbon source, cell growth and carbon flow through the Krebs cycle were greatly affected by the under-production of citrate synthase. In contrast, when glucose was the main nutrient, the same underproduction of citrate synthase had little effect on either growth or carbon flux. When the enzyme was overproduced 50-fold, cultures would grow on glucose but cell division could be abruptly stopped by adding acetate to the medium. These results indicate that the regulatory properties of citrate synthase are highly dependent on the carbon-source composition of the medium. Furthermore, recombinant DNA technology can be used to alter rate-controlling steps in biological pathways and elucidate the regulatory properties of metabolic systems.