In renal preservation, the longer the organ is cold stored the greater the damage to the organ. The mechanism of hypothermic-induced kidney injury is not known. In this study the effects of long-term preservation (up to 120 h) of the dog kidney on mitochondrial functions in an homogenate of kidney cortex tissue was investigated. Kidneys were exposed to either warm ischemia (0 to 90 min) cold ischemia (0, 72, 96, and J20 h). The mitochondrial oxygen uptake was measured in an homogenate. In both warm and cold ischemia there were changes in the mitochondrial utilization of oxygen. The changes were characterized as a decrease in uncoupler stimulated oxygen uptake by up to 40%, an increase in oligomycin-sensitive respiration by up to about 150%, and a decrease in the respiratory control ratio (uncoupler control ratio) from about 3 to 1. These changes in mitochondrial utilization of oxygen were partially reversed by including albumin in the respiration medium. Albumin binds free fatty acids and these may originate, during ischemia, from the action of phospholipases during ischemia. The changes in mitochondrial oxygen uptake may result from both the loss of membrane-bound phospholipids and the accumulation of free fatty acids. The changes in mitochondrial activity between 72 h (viable kidneys on transplantation) and 96 to 120 h preservation (nonviable kidneys) were not significant. Furthermore, reperfusion of kidneys preserved for 72 to 120 h resulted in a restoration of mitochondrial oxygen uptake to near normal (control) values. Thus, it does not appear that the limitation of successful long-term renal preservation is due to mitochondrial injury caused by cold ischemia.