Recently we reported that lymphotoxin (LT) administration protected non-obese diabetic (NOD) mice and BB rats from insulin-dependent diabetes mellitus. In this study we analysed the protection mechanism of LT by using cyclophosphamide (CY)-induced autoimmune diabetes in NOD mice. Pre-administration of 500 or 1000 U of LT three times a week between the age of 4 and 11-13 weeks before CY-treatment strongly inhibited CY-induced diabetes. This inhibition was reproduced by LT pre-administration at an earlier age (4 to 7 weeks) but not at a later age (8 to 11 or 10 to 12 wks). LT post-administration (100 U daily or 500 U twice a week) after CY-treatment at 14 weeks of age also strongly inhibited CY-induced diabetes. Spleen cell transfer was carried out using various combinations of donors and recipients. Spleen cell transfer from the non-diabetic mice, which were LT pre-administered between the age of 4 and 13 wks, to CY-treated mice did not significantly inhibit CY-induced diabetes, while transfer of the cells from the similarly treated mice to irradiated recipients did induce diabetes although the onset of diabetes was significantly delayed. Diabetes was not transferred by spleen cells from diabetic mice to LT pre-administered and CY-treated mice. LT administration did not change subpopulations and adhesion molecule expressions of the spleen lymphocyte. Taken together, these results suggest that LT protects NOD mice from CY-induced diabetes by making the mice resistant to autoimmune diabetes and possibly by suppressing anti-islet effector cells, but not by inducing adoptively transferable suppressor cells, although the precise mechanisms still remain to be elucidated.