Nalidixic acid was found to be most bactericidal against various species of gram-negative bacteria at 50 to 200 μg/ml. With all species tested, increases in the concentration of nalidixic acid above this range reduced, rather than increased, its bactericidal effect so that, at levels in the region of 400 μg/ml, the drug was relatively bacteriostatic. Therefore, the mode of action of nalidixic acid at various concentrations was investigated. It was found that at the most bactericidal concentration deoxyribonucleic acid synthesis, but no ribonucleic acid (RNA) or protein synthesis, was inhibited. However at higher concentrations, where the drug is least bactericidal, both RNA and protein synthesis were found to be inhibited. Results are presented which suggest that the protein synthesis inhibition is a secondary manifestation of the ability of the drug to inhibit RNA synthesis, and that of RNA synthesis is most likely the second target site for the action of the drug when bacteria are exposed to it in high concentrations. The clinical implications of these findings are discussed.