Sulfatide (3′-sulfogalactosyl-ceramide) is a glycosphingolipid mainly located in the nervous system, but has also been found in the islets of Langerhans. Previous studies have suggested that sulfatide is involved in insulin processing and secretion. In this study, sulfatide expression and metabolism in pancreas and isolated islets of the type II diabetes models, ob/ob- and db/db mouse, was investigated using TLC-ELISA, metabolic labelling and electron microscopy. As in non-diabetic Lewis rat and human pancreas, sulfatide was located in secretory granules of the beta cells. However, the type II diabetic animal models and their background strains had an altered sulfatide expression, involving the lack of the C16:0 sulfatide fatty acid isoform, compared to non-diabetic Lewis rat, BALB/c mouse and human pancreatic tissue, in which the two dominating pancreatic sulfatide isoforms C16:0 and C24:0 are expressed. Correspondingly, in isolated ob/ob islets, sulfatide synthesis excluded the production of C16:0 sulfatide. Insulin administration to ob/ob mouse, which lowers beta cell activity, resulted in significantly increased sulfatide expression in pancreas (p=0.0003), but still no expression of the C16:0 sulfatide isoform. In vitro, the C16:0 sulfatide was shown to be the isomer involved in the preservation of insulin crystals. Thus, it is hypothesized that the selection of sulfatide isomers in pancreas might be a genetic factor contributing to disease development in type II diabetic animal models.