The clinical significance of anti-pituitary antibodies (APA) was examined by enzyme-linked immunosorbent assay (ELISA) in individuals with non-insulin dependent diabetes mellitus (NIDDM). Serum samples were obtained from 150 NIDDM patients and 45 normal subjects. Urinary C-peptide (U- CPR) was also measured for the NIDDM patients. APA-positive serum was incubated with porcine pancreas, liver, kidney, or spleen powder and analyzed by immunoblot. The prevalence of APA was found to be significantly (P<0.05) higher in NIDDM patients (24.7%) than in the controls (6.7%) by ELISA. The index values for APA were inversely related to the levels of U-CPR (P<0.005). The levels of U-CPR were significantly (P<0.0001) lower and the prevalence of insulin deficiency was significantly (P<0.05) higher in NIDDM patients who were APA positive than in those who were APA negative. The presence of APA may therefore be related to reduced secretion of insulin in NIDDM patients. In Western blot analysis, preincubation of APA-positive sera with porcine pancreas powder prevented recognition of the 22-kD protein (APA). The possibility of a common autoantigenicity in the pancreas and the pituitary was indicated.