A rapid and sensitive method for the determination of cyclophosphamide (CP) and 5-fluorouracil (5-FU) and some of their metabolises in one analysis has been developed. Surface-coated open-tubular OV-275 columns were combined with electron-capture detection and nitrogen phosphorus selective detection. The influence of the column diameter on the separation is shown. Extraction with 2-propanol-diethyl ether (22:77) allows the isolation of CP, 5-FU and their analogues in one extraction step. The assay was applied to some pharmacokinetic experiments with chemotherapeutically treated patients and with a WAG/Rij rat.