We have utilized the NOD islet beta-cell line NIT-1 to monitor beta-cell specific autoantibodies and to investigate the modulation of IDDM in NOD mice by NIT-1 membrane associated antigens. The sera from diabetic but not from pre-diabetic or protected NOD mice strongly stained NIT-1 cells in FACS analysis. The cell surface antigens on NIT-1 cells were trypsin-sensitive. NIT-1 cells could not be stained by anti-mouse GAD67 antibody; however, we could demonstrate the presence of GAD65 and GAD67 mRNA by RT-PCR. Longitudinal analysis of anti-NIT-1 antibodies showed that these antibodies were present in the neonates but disappeared after weaning. Sonicated NIT-1 cell membrane preparations protected NOD mice from diabetes when injected intravenously in 5 week old mice. The protection was associated with reduced cytotoxic activity and elevated Th2-like responses as indicated by IgG1 antibodies against the NIT-1 cells. Subcutaneous injection of sonicated NIT-1 membranes or the injection of control red blood cell membranes failed to induce protection. We conclude that NIT-1 cell membranes do not express GAD but contain other antigens that are important in the development and prevention of IDDM. These antigens could be useful for the diagnosis of diabetes by monitoring autoantibody levels and for the modulation of IDDM by immunotherapy.