Induction of the peroxidase activity of cytochrome c (cyt c) by cardiolipin (CL) and H(2)O(2) in mitochondria is suggested to be a key event in early apoptosis. Although electrostatic interaction between the positively charged cyt c and negatively charged CL is a predominant force behind the formation of a specific cyt c-CL complex and sequential induction of the peroxidase activity, molecular mechanisms of hydrophobic interactions involving the fatty acyl chains of CL remain to be investigated. To elucidate the function of the acyl chains, particularly the role of the double bond, we synthesized a variety of CL analogues and examined their peroxidase inducing activity. Irrespective of the number of double bonds in the acyl chains, the peroxidase activity of cyt c induced by liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and a different CL (9:1 molar ratio) was similar, except for that of 1,1',2,2'-tetrastearoylcardiolipin (TSCL, C18:0)-containing liposomes. The peroxidase inducing activity of TSCL-containing liposomes was 3-4-fold greater than that of other CL-containing liposomes. The peroxidase activity induced by all CL-containing liposomes was much lower at high ionic strengths than that at low ionic strengths because of diminution of the electrostatic interaction. The peroxidase inducing effects of various CL-containing liposomes were related well to their ability to associate with cyt c. Thus, our results revealed that at low CL levels, the saturated acyl chain of CL is favorable for the activation of peroxidase activity of CL-bound cyt c and the proposed critical role of the double bond is not a general feature of the cyt c-CL interaction. The polarity of the membrane surface of TSCL-containing liposomes was slightly, but significantly, lower than that of other CL-containing liposomes, suggesting that the higher activating ability of TSCL-containing liposomes may be due to a reduced level of hydration of the polar head region reflecting tighter packing of the fully saturated acyl chains. Moreover, using CL analogues in which a central glycerol head moiety was modified, we revealed that the natural structure of the head moiety is not critical for the formation of the active cyt c-CL complex. The effects of the CL content of the liposomal membrane on the cyt c-CL interaction are discussed.