To study the role of CD27-CD27 ligand (CD27L)/CD70 interactions in the generation of murine allospecific T cell responses, SF9 cells or cell membranes expressing recombinant human CD70 were added to in vitro MLC containing C57BL/6 (H-2b) responder cells and class I and II MHC disparate H-2b/d stimulator cells. Alloantigen-specific CTL generation, CD8+ T cell proliferation, and levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase activity were enhanced in the presence of human CD27L/CD70 expressed on SF9 cell membranes. Enhancement of CD8+ T cell responses occurred in the absence of any discernible effects on CD4+ T cell proliferation or IL-2 responses. Additional studies demonstrated that CD27L/CD70-expressing membranes enhanced proliferative responses to class I MHC differences but had no effect on proliferative responses to class II MHC disparities. Enhancement of allospecific CTL generation was also observed when CD27L/CD70-expressing membranes were added only during the last 24 to 48 h of 5-day MLC. Thus, the present studies suggest that CD27-CD27L/CD70 interactions can selectively enhance differentiation of Ag-specific CD8+ T cell effector mechanisms under conditions in which Th cell responses are not altered.