Previous studies on the biliary secretion of inorganic mercury have concluded that inorganic mercury in bile is bound almost exclusively to substances of high molecular weight (HMW). In contrast, our results showed that inorganic mercury in bile is bound predominantly to a substance of low molecular weight (LMW), which is most likely glutathione (GSH). The previously reported binding of inorganic mercury to bile proteins is now explained as a postsecretory in vitro artifact resulting from the rapid oxidation of endogenous GSH which occurs during the collection and storage of bile samples. Gel filtration on Sephadex G-75 of freshly collected bile from rats treated with 203HgCl 2 or of control bile supplemented in vitro with 203HgCl 2 showed that most of the mercury was in the LMW fraction. On Sephadex G-25, the biliary mercury peak co-eluted with the mercury-GSH standard. However, when bile was allowed to stand at room temperature, there was a time-dependent shift of the mercury towards the HMW fraction. The rate of this shift was proportional to the rate of oxidation of GSH in bile. When GSH oxidation was inhibited by collecting bile in EDTA at 4°, the mercury remained associated with the LMW fraction. At a given GSH concentration in bile, the fraction of mercury bound to the HMW fraction was independent of mercury concentration, in the range of 0.05 to 5.0 μM HgCl 2. These results suggest that the inorganic mercury was secreted into bile complexed with a LMW substance. This LMW substance has been tentatively identified as GSH.