Purpose. We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-α. Here, we hypothesize that BSA-induced increase in the release of TNF-α, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism. Methods. Rat livers were perfused ex vivo for 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n = 4-5/group). At the end of perfusion, liver microsomes were prepared and analyzed for their total CYP, CYP2E1, CYP3A2, and CYP2C11 protein contents and the activities of cytochrome c reductase, CYP2E1, CYP3A2, CYP2C11, CYP2E1, CYP2D1, CYP1A1, and CYP2B1/2. In addition, the concentrations of various cytokines and nitric oxide were quantified in the outlet perfusate. Results. In the absence of BSA, the perfusate levels of all measured cytokines and nitric oxide were low. However, when the perfusate contained BSA, the levels of TNF-α, interleukin-6, and nitric oxide increased significantly (p < 0.005). Perfusion of the livers for 3 h with the BSA-containing perfusate resulted in significant (p < 0.05) decreases in the total CYP (41%), CYP2E1 (59%), CYP3A2 (68%), and CYP2C11 (50%) protein contents and activities of cytochrome c reductase (31%), CYP2E1 (66%), CYP3A2 (54%), and CYP2C11 (51%). In contrast, perfusion of livers for 1 or 2 h with the BSA perfusate did not have any significant effect on CYP-mediated metabolism. The CYP1A2, CYP2D1, and CYP2B1/2 activities were not affected by BSA, regardless of perfusion time. Conclusion. Addition of BSA to perfusates, which is a routine practice in isolated rat liver studies, can reduce CYP-mediated drug metabolism by a mechanism independent of protein-binding effect.