Mutants of Escherichia coli K12, defective in phosphatidylserine synthetase (pss), can be isolated as temperature-sensitive, conditional lethals. When cultivated at intermediate temperatures (30 degrees), such mutants contain approximately 3 times more phosphatidylglycerol plus cardiolipin (and less phosphatidylethanolamine) than normal. We now wish to report that, under these conditions, the pss-8 mutant is hypersensitive to certain antibiotics, especially to streptomycin, kanamycin, and gentamicin, although also to ampicillin and novobiocin. At 30 degrees, the membrane protein and fatty acid composition of pss-8 is nearly normal, i.e. identical with an isogenic pss+ organism. Radiochemical labeling and bacteriophage growth studies show that lipopolysaccharide is also unaltered. Therefore, the antibiotic hypersensitivity of pss-8 differs from previously reported hypersensitivities, associated with lipopolysaccharide defects. These results suggest that the polar phospholipid headgroups may play an important role in maintaining the barrier function of the outer gramnegative membrane and that putative inhibitors of the phosphatidylserine synthetase might potentiate the action of numerous antibiotics currently in clinical use.