The specific lysis of bloodstream trypanosomes by serum from a nonpermissive mammalian host is the result of interaction between the serum trypanocidal factor (high-density lipoprotein) and the trypanosome surface. The studies described in this paper attempt to define further the mode of action of this cytotoxic lipoprotein. The binding of high-density lipoprotein to Trypanosoma brucei was instantaneous at 4 degrees C and readily reversible. Binding was not mediated by the surface glycoprotein as removal of the surface coat enhanced binding at 4 degrees C, and no stable glycoprotein-lipoprotein complex could be detected. Pretreatment of trypanosomes with the cross linker dimethylsuberimidate rendered cells resistant to lysis. Addition of membrane-stabilizing drugs, such as cytochalasins C, D, and E, and local anesthetics (dibucaine, tetracaine, and procaine), also inhibited high-density lipoprotein-induced cell lysis. The data presented support the idea that at 37 degrees C lateral diffusion of the variant surface glycoprotein, an integral membrane protein, allows maximal high-density lipoprotein-cell interaction in serum-sensitive cells, and that altered properties of the plasma membrane induced by low temperature or the addition of cytochalasins, local anesthetics, or zinc inhibit this interaction, possibly by increasing the shielding of the plasma membrane by more rigidly anchored surface glycoprotein molecules.