Multiple effector cells have been implicated in transplant rejection, including cytotoxic T cells, B cells, macrophages and NK cells. The purpose of this study was to examine the effector pathways which are critical to murine cardiac allograft rejection. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis of syngeneic and allogeneic vascularized heterotopic cardiac grafts at 5, 8 and 12 days following transplantation demonstrate constitutive expression of Fas in both the syngeneic and allogeneic grafts as well as in normal heart. However, FasL, granzyme, and perforin expression were shown to be up-regulated on days 5-12 in the allograft with no expression in syngeneic grafts or in normal hearts. We have recently analyzed the functional significance of T cell cytotoxic pathways and found that neither the Fas nor CD8+ cytotoxic pathways are required for murine cardiac allograft rejection. In light of these results, we investigated the functional significance of other effector cells in the rejection process. B cell deficient C57BL/10-IgHtm1Cgn mice rejected cardiac allografts from normal donors at control rate. Finally, RT-PCR was used to analyze the expression of macrophage effector transcripts in allograft rejection. Transcripts for iNOS (inducible nitric oxide synthase) and TNF alpha (tumor necrosis factor-alpha) were up-regulated on days 5-12 in untreated allografts with undetectable expression in normal heart or syngeneic grafts. These results demonstrate that effective allograft rejection can occur in the absence of B cells and T cell cytotoxicity pathways suggesting that other effector pathways, such as delayed-type hypersensitivity responses by macrophages, may be critical for allograft rejection.