Neurotropin (NSP) is an extract isolated from vaccinia virus-inoculated and inflamed skin of rabbits. The present study was undertaken to examine the immunostimulating actions of NSP. NSP alone did not induce a proliferation of spleen cells from mice or human peripheral blood lymphocytes (PBL). However, NSP clearly enhanced concanavalin A (Con A)-induced proliferation of both spleen cells and human PBL, but slightly inhibited lipopolysaccharide (LPS)-induced proliferation of spleen cells. NSP reversed the decreases of Con A-induced proliferation of spleen cells pretreated with mitomycin C (MMC) in a dose of 5 microgram/ml or irradiated at 100 to 500 R, while NSP did not reverse the decreases of LPS-induced proliferation of spleen cells pretreated with MMC, in a dose of 5 to 50 microgram/ml, or irradiated at 100 to 1000 R. NSP enhanced in a dose-dependent fashion one-way mixed lymphocytes culture (MLC) reaction using MMC-treated spleen cells from BDF1 mice as stimulator cells and those from C57BL/6 mice as responder cells, respectively. NSP reversed the considerable decrease of MLC-mediated proliferation of responder cells pretreated with MMC, in a dose of 5 to 10 microgram/ml, or irradiated at 100 to 250 R. These results indicate that NSP activates T cell function participating in cell-mediated immunity.