The purpose of this study was to determine whether the effect of arachidonic acid on hepatic O2 uptake is O2 dependent and which region of the liver lobule it affects. In livers perfused at normal flow rates, infusion of arachidonate increased O2 uptake significantly by about 20-25 mumol.g-1.h-1. When the flow rate was doubled to make the hepatic O2 gradient shallower, the increase in O2 uptake due to arachidonate was two to three times larger (i.e., approximately 50 mumol.g-1.h-1). In livers perfused in the retrograde direction, maximal rates of O2 uptake were about twofold higher in upstream pericentral than in downstream periportal regions, and arachidonic acid nearly doubled O2 uptake in downstream areas without affecting rates in upstream regions. Thus it is concluded that arachidonate stimulates O2 uptake in an O2-dependent manner. This effect was sensitive to an inhibitor of the lipoxygenase, nordihydroguaiaretic acid, in perfused liver but not in isolated hepatocytes. In addition, conditioned medium from Kupffer cells incubated at high O2 tension stimulated parenchymal cell O2 uptake. Furthermore, arachidonate increased intracellular Ca2+ in isolated Kupffer cells in a dose-dependent manner. These findings suggest that eicosanoids produced by nonparenchymal cells participate in a hepatic O2 sensor mechanism involving Ca2+ that regulates O2 uptake by parenchymal cells in the liver.