Endotoxemia results in both the down-regulation of multiple cytochrome P450 genes and the induction of inducible nitric oxide synthase (NOS2). The nitric oxide (NO) released during inflammation has been implicated as the mediator of the decreased catalytic activity and expression of several cytochrome P450 isozymes. We examined the role of NO in the decreases in both gene expression and activity of three P450s in endotoxemic parental and NOS2 knockout mice. Twenty-four hours of endotoxin (LPS) treatment significantly suppressed CYP2C29 and CYP3A11 mRNA expression in both the parental and NOS2 knockout strains. Microsomal CYP2E1, CYP2C-like, and CYP3A-like protein levels were also decreased in both strains of mouse. Similar results were obtained in parental strain endotoxemic mice co-administered the NOS inhibitor aminoguanidine. Six hours after LPS treatment, there was an NO-dependent decrease in testosterone 6beta-hydroxylase activity, because no decreases in activity were observed in the NOS2 knockout mice or in mice co-administered aminoguanidine. LPS also evoked decreases in testosterone 15alpha- and 16beta-hydroxylase activity after 24 hr that were observed in the parental strain and not in NOS2 knockout mice. Our results demonstrate that the down-regulation of CYP2C-like, CYP3A-like and CYP2E1 proteins and mRNAs, in the endotoxemic mouse can occur independently of NO production. We do, however, show that the NO released during endotoxemia is capable of causing decreases in some cytochrome P450 catalytic activities.