At a concentration of 2.5 mM, dl-glyceraldehyde 3-phosphate has a bactericidal effect upon Escherichia coli. The glycerol 3-phosphate transport system is required for the entry of the biologically active l-enantiomer. l-Glyceraldehyde must be phosphorylated by the cell to exert its full effect upon growth. The addition of dl-glyceraldehyde 3-phosphate to a culture of E. coli caused no preferential inhibition of the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although protein accumulation was less affected. Studies with mutant strains ruled out catabolic glycerol 3-phosphate dehydrogenase, anabolic nicotinamide adenine dinucleotide (phosphate):sn-glycerol 3-phosphate oxidoreductase, and fructose 1,6-diphosphate aldolase as the primary sites of action. l-Glyceraldehyde 3-phosphate is a competitive inhibitor of sn-glycerol 3-phosphate in the reactions catalyzed by acyl coenzyme A:sn-glycerol 3-phosphate acyltransferase (K(i) of 1.8 mM) and cytidine 5'-diphosphate-diglyceride:sn-glycerol 3-phosphate phosphatidyltransferase (K(i) of 2.7 mM). A K(m) mutant for the former enzyme was susceptible to the inhibitor. l-Glyceraldehyde 3-phosphate does not affect acyl coenzyme A:lysophosphatidate acyltransferase activity. In vivo, phosphatidylethanolamine and phosphatidylglycerol accumulation are inhibited to the same extent by the addition of dl-glyceraldehyde 3-phosphate to a culture of E. coli.