The analysis of phosphoramide mustard by reversed-phase ion pair high pressure liquid chromatography using ultraviolet detection is described. The method will enable measurement of serum concentrations of phosphoramide mustard over a period of approximately 24 h after an i.v. dose of 60 mg/kg of cyclophosphamide and requires 0.5 ml of serum or plasma. The between-day precision of the assay at concentrations of 0.34, 2.76, and 6.90 mg/L gave coefficients of variation of 16, 10, and 9%, respectively. The percentage analytical recovery of phosphoramide mustard is 35%. Preliminary results providing pharmacokinetic data on two adults undergoing high-dose cyclophosphamide treatment prior to bone marrow transplant are presented.