The effects of administration of the cytotoxic agent cyclophosphamide on cytochrome P450 have been examined in the liver microsomes of male rats. Microsomes were prepared after cyclophosphamide administration 1, 4 or 7 days prior to killing. The coadministration of cyclophosphamide with N-acetylcysteine has also been investigated. The microsomes were assayed for NADPH cytochrome P450 reductase, aminopyrine demethylase, erythromycin demethylase and androstenedione hydroxylase activities. Activities were generally unchanged 1 and 4 days after cyclophosphamide administration and were significantly decreased at 7 days. N-Acetylcysteine did not alter the effects of cyclophosphamide at 7 days. The effect of cyclophosphamide in vitro has also been examined. Microsomes from untreated animals were subjected to the above assays following in vitro metabolic activation of cyclophosphamide in a reconstituted system in the presence and absence of N-acetylcysteine. All enzyme activities were significantly reduced by the cyclophosphamide metabolites. The presence of N-acetylcysteine prevented this inactivation. The results of these investigations suggest that cyclophosphamide inactivates hepatic cytochrome P450 in vitro and in vivo via different mechanisms.