Phosphoramide mustard, an active metabolite of cyclophosphamide, has been reacted separately with guanosine and deoxyguanosine in aqueous solution at pH 7.4. The major adduct which was formed in each case has been isolated by reverse-phase high-pressure liquid chromatography. The structure of the major adduct, as determined by a combination of ultraviolet and field desorption mass spectrometry, is that of phosphoramide mustard, one arm of which has reacted with guanosine or deoxyguanosine in position 7. These adducts are much less stable than was 7-methylguanosine, and they decompose with a half-life of 1.3 hr at 37 degrees and pH 7.4. This instability may contribute to the action of phosphoramide mustard at a molecular level.