In the present study we have examined the potential role of xanthine oxidase (XO) in the intracellular oxidative stress induced by combinations of recombinant murine TNF alpha (rMuTNF alpha) and murine interferon-gamma (IFN gamma) in cultured mouse hepatocytes. IFN gamma alone and the combination of rMuTNF alpha and IFN gamma increased XO activity after a 4 hr exposure period. rMuTNF alpha alone increased XO activity only after 24 hr. At the later time point, the increased XO activity was accounted for by decreased XDH activity. However, the apparent conversion of XDH to XO cannot account for the early effects of rMuTNF alpha on hepatocyte function, particularly the onset of an oxidative stress (as indicated by efflux of GSSG from the hepatocytes). This effect is observed after two hours, and it is temporally the earliest sign of alteration of cellular function caused by rMuTNF alpha. Increased XO activity was not observed until 4 hr after treatment with rMuTNF alpha/IFN gamma. In addition, inhibition of XO activity with allopurinol did not ameliorate GSSG efflux from hepatocytes treated with the cytokines. However, the ATP depletion caused by the combination of rMuTNF alpha and IFN gamma and the cytotoxicity observed with the combined cytokines in cells pre-treated with BCNU (to inhibit glutathione reductase) was inhibited by allopurinol. These results show that the onset of oxidative stress in cultured mouse hepatocytes is not due to conversion of XDH to XO. However, events which follow the efflux of GSSG, such as ATP depletion and cytotoxicity in cells with impaired anti-oxidant defenses, may be partially due to increased XO activity, especially in cells treated with both rMuTNF alpha and IFN tau.