The aim of the present study is to investigate the influence of the environmental factors, smoking and alcohol, on the biotransformation of cyclophosphamide (CP) in the rat in vivo and in vitro with S9 liver fractions. The biotransformation of CP was studied by the determination of the CP metabolites, nor-nitrogen mustard (NNM), 4-ketocyclophosphamide (KCP), and carboxyphosphamide (CAR). The effect of the environmental factors, smoking and alcohol consumption, on the biotransformation enzymes was mimicked by pretreatment of rats with beta-naphthoflavone and ethanol, respectively. Rats treated with olive oil and water served as controls and rats pretreated with Aroclor 1254 and phenobarbital were used as positive controls. The influence of sex and supplementation with NAD and GSH, mimicking a biological variation in NAD and GSH levels in rat and human liver, was also studied. Pretreatment of rats with Aroclor 1254 decreased the excretion of unmetabolized CP in urine, most likely due to an enhanced biotransformation. The in vitro hepatic biotransformation of CP in rats was strongly influenced by sex, by supplementation with NAD and GSH, and by pretreatment with the enzyme-inducers, phenobarbital and Aroclor 1254. No influence of pretreatment with the enzyme-inducers, beta-naphthoflavone and ethanol, was found. The results suggest that the influence of the environmental factors, alcohol consumption and smoking, on the biotransformation of CP in man will be negligible.