Previous studies showed that islet cell autoantibodies are present at the onset of insulin-dependent diabetes mellitus (IDDM) in humans and in rodent models of this disease. The targets of these antibodies are not well characterized. Using an immunoblot assay on protein extracts from rat insulinoma (RIN) cells (RINm5F), we showed that serums from nonobese diabetic (NOD) mice bind to a 52,000-Mr islet cell antigen. Nondiabetic NON mice do not have antibodies to this antigen. The NOD and NON serums also contained antibodies to salivary gland proteins. Analysis of the tissue distribution of the 52,000-Mr antigen revealed that it is present in purified RINm5F membranes but is not found in other endocrine or nonendocrine tissues. Autoantibodies to an antigen of similar molecular weight are detected in 29% of human IDDM serums. To determine whether the autoantibodies from mouse and human serums bind the same antigen, two-dimensional immunoblots were carried out. The 52,000-Mr protein isoforms appeared identical when probed with NOD and human IDDM serums. We conclude that serums from NOD mice and some humans with IDDM contain similar autoantibodies to a 52,000-Mr RINm5F cell-specific membrane protein. The presence of autoantibodies to this 52,000-Mr islet cell protein at the onset of the disease suggests that it may be an important antigen in IDDM.