Preservation of the liver involves a period of cold (0° to 4°C) ischemia; the longer the ischemic period, the greater the injury to the liver. The mechanisms for cold-induced ischemic injury are not known, but it is clear that after preservation the liver has a reduced capacity to regenerate high-energy phosphate compounds (ATP). One cause for the delayed rate of ATP synthesis could be injury to the mitochondria. The effects of long-term (more than 24 hr) preservation on liver mitochondrial function have not been previously studied. In this study, rat livers were preserved in University of Wisconsin solution at 4° C for up to 96 hr. After preservation, mitochondrial respiratory function was assayed in a homogenate and in isolated mitochondria. We saw a progressive increase in oligomycin-sensitive respiration with time of preservation (from 1.2 ± 0.09 m̈mol · min−1 · gm tissue−1 at 0 hr to 3.8 ± 0.2 m̈mol · min−1 · gm tissue−1 after 96 hr). The increase after 24-hr preservation (2.1 ± 0.2 m̈mol · min−1 · gm tissue−1) was also significantly greater than 0 time values (p < 0.05). No decrease was found in uncoupler-stimulated respiration for up to 48 hr of preservation; only a small decrease was seen after 72 hr of preservation (about 30%). The cause of the increase in oligomycin-sensitive respiration appeared to be related to free fatty acids (or another uncoupling factor) generated during preservation. This was suggested from the fact that bovine serum albumin prevented the increase in oligomycin-sensitive respiration after all periods of preservation. The increase in oligomycin-sensitive respiration suggests an uncoupling of mitochondrial respiratory functions that could lead to a decreased rate of resynthesis of ATP on transplantation of the liver. Chlorpromazine and verapamil also prevented the increased oligomycinsensitive respiration caused by preservation, suggesting that calcium-activated phospholipases contributed to the generation of free fatty acids (or other factors) that caused mitochondrial injury. Rat livers flushed out with Ringer's lactate containing bovine serum albumin after 48 hr of preservation showed improved mitochondrial function, and no increase was found in oligomycin-sensitive respiration. This suggests that before transplantation the liver should be flushed out with a solution containing albumin to remove the uncoupling factors and improve mitochondrial function. (HEPATOLOGY 1992;16:742–748.)