Perforin is a 68 kD protein found in the granules of cytotoxic lymphocytes and is used by lymphocytes to form lethal pores in the membranes of the cells they kill. We and others have found that when perforin is purified, its lytic activity is markedly reduced. ELISAs indicated that our final recovery of perforin protein was excellent. We decided to determine if depletion of other granule proteins contributed to the loss of lytic activity. We isolated perforin to the point where lytic activity was diminished and added back granule proteins that had no lytic activity or detectable (antigenic) perforin. Perforin was isolated by Cu2+-immobilized metal affinity chromatography (IMAC) followed by phenyl-Superose hydrophobic interaction chromatography (HIC). Its lytic activity was enhanced by a low molecular weight (<15 kD) protein, perforin enhancing protein (PEPr). We have isolated PEPr by two methods, HIC and MonoQ. Nonlytic PEPr restored perforin to close to its original lytic activity. A protein similar if not identical to PEPr was also detectable as an 125I-labeled protein associated with lytic perforin. We propose that PEPr acts in conjunction with perforin to form lethal pores and suggest that PEPr may be the rat equivalent of the human cytotoxic lymphocyte protein, granulysin.