Immuno-electron microscopy technique was employed to investigate the cellular distribution of 60 kDa heat-shock protein (HSP60) in pancreatic Beta cells of control and non-obese diabetic mice. In thin sections prepared from control mice, antibody to mammalian HSP60 cross-reacted with protein(s) located to mitochondria and secretory granules. In particular, prominent binding of the antibody was seen to the insulin core of the mature insulin-secreting granules. In comparison, very little immunoreactivity was observed with immature secretory granules or with the Golgi apparatus. No binding to secretory granules or mitochondria was observed with normal mouse serum or with unrelated sera. On Western blots, HSP60 antibody specifically interacted with a single 62 kDa islet cell protein. These results suggest the existence of an HSP60-related protein with a novel location in mature secretory granules of Beta cells. The preferential association of the HSP60-related protein with the insulin core was gradually lost in Beta cells of pre-diabetic non-obese diabetic mice, and correlated with the progression of insulitis. The decrease in the granular binding of the HSP60 antibody was accompanied by an increase in cytoplasm staining, and was concomitant with a significant expansion of the insulin core diameter. The altered distribution of the HSP60-related protein in prediabetic mice, together with our observation that immature secretory granules accumulate in these animals indicate that the presence of HSP60-related protein in secretory granules might be associated with the secretory function of Beta cells.