Background. Our previous studies have shown that the ability of donor bone marrow to augment skin graft survival in antithymocyte serum (ATS)- treated recipients is dependent on the presence of functional CD95-ligand (Fas-ligand) molecules on donor cells. Because donor spleen cells can augment graft survival to a similar degree in the same model, we investigated whether the donor Spleen cell effect was also dependent on the presence of CD95- ligand on donor cells and CD95 on recipient cells. Methods. Mutant mice bearing defects in the expression of CD95 (lpr mutation) and CD95-ligand (gld mutation) were used as recipients and cell donors, respectively. Recipients were injected with rabbit ATS on days -1 and +2, and then were injected with 5x107spleen cells on day +7. Skin graft survival was compared and correlated with the use of mutant mice as recipients and cell donors. Results. The combination of ATS and infusions of wild-type [median survival (MST)=44 days, P=0.0004] and g/d (mutant CD95-ligand, MST=37 days, P=0.02) donor spleen cells enhanced C3H graft survival, compared with (C57BL/6 x A)F1 recipients treated with ATS alone (MST=27 days). Furthermore, C57BL/6 lpr (CD95-deficient) strain recipients treated with ATS and donor spleen cells demonstrated enhanced B10.D2(R107) strain skin graft survival (MST=44 days, P=0.003), compared with C57BL/6 lpr recipients treated with ATS alone (MST=31 days). Wild-type C57BL/6 recipients treated in the same manner also exhibited an extension of graft survival (MST=64 days) versus controls treated with ATS alone (MST=31 days). Conclusion. The data demonstrate that the ability of donor spleen cells to augment allograft survival is not dependent on the CD95/CD95-ligand pathway; therefore the deletion of allospecific cells by donor spleen cells may be induced via a pathway other than deletion by donor bone marrow cells.