To study the effects of fatty acids and ketone bodies on both insulin release and lasting changes in ultrastructure and function of the pancreatic B-cell, isolated mouse islets were cultured for one week in the presence of either octanoate, acetoacetate or 3-hydroxybutyrate. The glucose concentration of the unsupplemented culture medium was 3.3 mmol/l and islets cultured in this medium were used as controls. It was found that octanoate stimulated insulin accumulation in the culture medium, decreased the islet insulin content and also effected a degranulation of the islet B-cells. Although there was no significant change in the total mitochondrial volume of the octanoate cultured islets, the activity of the mitochondrial marker enzyme L-3-hydroxyacyl-CoA-dehydrogenase was decreased. In short-term incubations performed after culture with octanoate, there was an increased rate of islet protein biosynthesis, which seemed to be independent of a glucose challenge. After culture of islets with acetoacetate, morphometrical measurements indicated an increased islet cell size, but there appeared to be no further effects of this ketone body on the islet ultrastructure or function. 3-Hydroxybutyrate, however, increased both the insulin accumulation in the culture medium and the insulin content of the cultured islets. Apparently, the β-granule content of the islets exposed to 3-hydroxybutyrate also increased. Furthermore, there was a reduction in the total mitochondrial volume of the 3-hydroxybutyrate cultured islets. In addition, the activity of L-3-hydroxyacyl-CoA-dehydrogenase was reduced, whereas islet glucose oxidation was markedly increased. In conclusion, therefore 3-hydroxybutyrate greatly resembled glucose in its effects on the islet B-cells.