Recent evidence has implicated phosphoramide mustard as a highly active metabolite of cyclophosphamide (CP). This compound has been shown to be produced from CP both in vitro and in vivo. To further study the role of this active metabolite we have devised methods to quantitate the compound in human serum and urine using gas chromatography-chemical ionization mass spectrometry. Tetradeuterated phosphoramide mustard has been synthesized and is used as an internal standard. CP and nor-nitrogen mustard have also been quantitated in human samples using analogous tetradeuterated internal standards.