The alkylating agent cyclophosphamide (CPA), widely used in paediatric oncology, is a prodrug which requires activation by the hepatic cytochrome p450 (CYP) enzyme system to form the cytotoxic metabolite phospharamide mustard. However, the drug can also be metabolized to form a number of inactive metabolites which do not have anti-tumor activity. In a recent study investigating the use of CPA for the treatment of B-Cell non-Hodgkin lymphoma (NHL), it was reported that relapsed patients had higher blood concentrations of the inactive metabolites and a lower rate of clearance from the blood of the parent drug. The significant variations in metabolism observed among individuals may be related to the various functional single nucleotide polymorphisms (SNPs) that have been reported in the CYP enzyme system. In an ongoing study we have investigated the pharmacokinetics of CPA in a total of 29 children with B-Cell NHL or soft tissue sarcoma. A liquid chromatography-mass spectrometry assay was developed to estimate the plasma concentrations of CPA and three inactive metabolites. Non-compartment analysis was used to estimate the clearance of CPA, which was found to correlate inversely with concentrations of the inactive metabolites.