The intracellular transformation of cis-mafosfamide has been studied in P388 mice leukemia cells using 31P-NMR spectroscopy. For this purpose the cells were entrapped in low-gelling-temperature agarose threads. Internal pH of the cells, determined from the position of the intracellular inorganic phosphate, was 7.2. The cell membrane was permeable to 4-hydroxycyclophosphamide and aldophosphamide and less permeable to phosphoramide mustard. 4-Keto-cyclophosphamide and carboxyphosphamide signals were not detectable in cells either sensitive or resistant to oxazaphosphorine treatment.