Escherichia coli beta-alanine auxotrophs (panD2) were used to manipulate the specific cellular content of coenzyme A (CoA) and assess the associated physiological effects. Growth-limiting concentrations of CoA resulted in an increase in phospholipid/protein ratio in relA1 mutants, but not in their rel+ counterparts, indicating that protein biosynthesis was more severely affected by CoA deprivation than phospholipid biosynthesis. Acetyl-CoA was the dominant component (79.8%) of the CoA pool in cells exponentially growing in glucose-minimal medium, with significant concentrations of CoA (13.8%) and succinyl-CoA (5.9%) also detected. Malonyl-CoA was a minor species (0.5%), and the mixed disulfide of CoA and glutathione was not present. Acetyl-CoA was also the major constituent in cells depleted of CoA. On the other hand, succinyl-CoA was absent, suggesting that the protein synthesis defect may be due to the inability to generate sufficient quantities of precursors via the tricarboxylic acid cycle to support amino acid biosynthesis. Production of acyl carrier protein was controlled in part by the availability of CoA, and the lower concentration of acyl carrier protein in CoA-depleted cells was associated with a concomitant decrease in the saturated/unsaturated fatty acid ratio.