When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor (5,12). Intracellular L-GAP can also be formed by a glycerokinase-catalyzed phosphorylation of L-glyceraldehyde that is taken up from the growth medium (12). The mechanism by which L-GAP causes E. coli to lose viability is not known.One possibility is that L-GAP exerts its bactericidal effect by interfering with a critical biochemical reaction. In aqueous solution, L-GAP exists as an equilibrium mixture of the hydrated and free aldehyde in a molar ratio of 29:1 (14). Since the hydrated form may be considered an analog of sn-G3P, L-GAP has the potential to interfere with enzymes involved in sn-G3P metabolism. L-GAP is a competitive inhibitor of both sn-G3P acyltransferase and phosphatidylglycerol phosphate synthetase (12, 13). However, these inhibitory activities do not necessarily explain the bactericidal activity of L-GAP. The sn-G3P analogs 3-hydroxy-4-oxobutyl-1-phosphonate and 1,3,4-trihydroxybutyl-1-phosphonate are bacteriostatic rather than bactericidal, even though they also inhibit the two lipid enzymes (13; unpublished