The kinetics of nalidixic acid (NA) and its two metabolites, 7-hydroxynalidixic acid (HNA) and 7-carboxynalidixic acid (CNA) were studied after a single oral dose and after two doses a day for 7 days. Total unconjugated NA, HNA, and CNA concentrations in plasma and urine samples were assayed by a new high-pressure liquid chromatographic (HPLC) technique. NA was rapidly absorbed and hydroxylated to HNA. Both were rapidly excreted in urine with an apparent elimination half-life (t1/2) of 6 to 7 hr. CNA was never detected in the plasma, although it accounted for about 25% of total urinary NA and metabolites. No major sex-related differences in the kinetics and the metabolic pattern were observed. During the 7-day treatment there was no significant cumulation of NA and HNA. The study demonstrated that the concentration of the active compounds, unconjugated NA and HNA, was more than five times the minimum bacteria inhibiting concentration for 8 hr after drug and slightly above this concentration during the next 4 hr.