Reconstituted membranes consist of liposomal structures formed by removal of detergent from solubilized membrane constituents. The membrane-like configuration of reconstituted membranes makes them attractive as vehicles for presentation of tumor-associated antigens and induction of immune responses. In this study the potential of immunomodulators was assessed to enhance the specific immune response induced by immunization with reconstituted membranes prepared from SL2 lymphosarcoma cells. Reconstituted membranes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) provided better protection against a challenge with SL2 cells than did reconstituted membranes containing alternative immunomodulators. Local administration of IL-2 at the immunization sites further augmented the protection induced by reconstituted membranes with MTP-PE, but was ineffective when administered with plain reconstituted membranes. Immunity elicited by the triple modality of reconstituted SL2 membranes with MTP-PE and IL-2 was specific for SL2 cells. Systemic immunity was obtained against a challenge with a 100-fold higher number of SL2 cells than was reached after immunization with reconstituted membranes alone (10(5) vs. 10(3) SL2 cells). Macrophages isolated from the peritoneal cavity of immunized mice 5 to 7 days after tumor challenge expressed high in vitro cytotoxicity. However, in contrast to the observed specificity of the systemic immunity, macrophages killed both SL2 cells and non-related P815 cells. Neither major cytotoxic lymphocyte activity nor substantial cytotoxic antibody titers were detectable. These results clearly indicate that the approach using reconstituted membranes combined with particular immunomodulators warrants further exploration for the development of safe, well-characterized cancer vaccines.