The antibiotic combination of sulfamethoxazole and trimethoprim was evaluated for treatment of peritonitis in patients in renal failure undergoing continuous ambulatory peritoneal dialysis. Although current methods of analysis were adequate for measurement of sulfamethoxazole, a review of the available methods of analysis for trimethoprim did not yield a satisfactory method. Therefore, a high performance liquid chromatography (HPLC) assay was developed to follow the pharmacokinetics of trimethoprim in serum and peritoneal dialysate fluid. In this assay, trimethoprim is extracted from plasma, serum, or dialysate fluid by solid-phase column chromatography that is efficient (82% recovery), quick, and simple to use. The HPLC method utilizes a common reverse-phase system with a 0.01 M sodium acetate and acetonitrile mobile phase and detection at 254 nm. The assay offers excellent between-run replication (p = 0.96), high sensitivity (0.05 microgram/mL), and linearity over a wide range (2-100 micrograms/ml; r = 0.99). The method offers freedom from interference by metabolites and a wide range of commonly administered drugs. It is suitable for other pharmacokinetic studies involving trimethoprim but not its metabolites, and also for clinical assay of trimethoprim in situations where high levels of the antibiotic are necessary to combat resistant organisms and in serious infections by opportunistic organisms such as Pneumocystis carinii.