Facilitation of fatty acid uptake in beta cells could potentially affect beta cell metabolism and secretory function; however such effects have not been clearly documented. CD36 facilitates uptake of fatty acids (FA) in muscle and adipose tissue and is likely to exert a similar effect in beta cells. We investigated the impact of over-expressing CD36 on fatty acid uptake and beta cell function by a Tet-on system in INS-1 cells. Doxycycline dose-dependently increased the CD36 protein with localization mainly in the cell membrane. Over-expression increased both specific uptake and efflux of oleate whereas intracellular glycerides were only marginally increased and incorporation of 14C-oleate or -palmitate into di- or triglycerides not affected. The normal potentiation of glucose-induced insulin secretion by acute addition of FA (50-100 micromol/l oleate and palmitate) was lost and the normal inhibitory effect of high glucose both on oleate oxidation and on the activity of carnitine palmitoyltransferase I was reduced. Over-expression did not induce apoptosis. We conclude that induction of the CD36 transporter increases uptake of FA, the consequences of which are blunting of the functional interplay between glucose and FA on insulin secretion and oxidative metabolism.