We developed a high-performance liquid chromatographic assay to measure ceftriaxone in serum, urine, and cerebrospinal fluid. Ion pairing was used because ceftriaxone is a relatively polar compound which is poorly retained on C18 columns in standard reverse-phase high-performance liquid chromatography and which produces trailing peaks in the absence of ion-pairing agents. The mobile phase was a combination of acetonitrile and water (46:54), adjusted to pH 9.0 with 10 mM K2HPO4, which contained 10 mM hexadecyltrimethylammonium bromide as the ion-pairing agent. Moxalactam (200 micrograms/ml) was used as the internal standard. A silica-packed precolumn (3 cm long) was used to prevent rapid deterioration of the analytical column (30 by 0.4 cm) by the alkaline pH of the mobile phase, and it significantly extended the life of the analytical column. The assay was linear with ceftriaxone concentrations of 1 to 250 micrograms/ml (r = 0.999) and correlated well with an agar diffusion bioassay (r = 0.990). Reproducibility was good, with intrarun coefficients of variation from 2.3 to 6.4% and interrun coefficients of variation from 3.2 to 21.4%. The absolute recoveries of ceftriaxone and moxalactam were 91 to 97 and 96 to 98%, respectively. No interferences were observed with more than 40 commonly prescribed drugs, including 10 cephalosporins (cefotaxime, cefoperazone, ceftazidime, ceftizoxime, cefoxitin, cefamandole, cephalothin, cefazolin, cephapirin, and cephalexin), or with sera from patients with renal or hepatic disease.