In vitro and in vivo studies in animals have shown that elevated levels of free fatty acids (FFAs) induce impaired beta-cell function corresponding to the abnormalities observed in non-insulin-dependent diabetes mellitus (NIDDM). Previously, it was demonstrated that the chain length and degree of unsaturation are of importance for the insulinotropic effect of fatty acids. However, it is not known if the spatial configuration of the fatty acid influences beta-cell function. The present study examines whether cis and trans fatty acids acutely influence insulin release and glucose oxidation in isolated mouse islets in the same way and to the same extent. Thus, we studied the impact of both cis and trans forms of C 18:1 fatty acids. We found that cis and trans vaccenic acid (cis and trans C 18:1 delta11), as well as oleic acid (cis C 18:1 delta9) and elaidic acid (trans 18:1 delta9), caused a dose-dependent increase in glucose (16.7 mmol/L)-stimulated insulin secretion during static islet incubations. The maximal stimulatory effect for cis and trans vaccenic acid and for oleic and elaidic acid was observed at concentrations of 2.0 and 3.0 mmol/L, respectively. The trans isomers, trans vaccenic and elaidic acid, elicited a higher maximal insulin output than the respective cis isomers, cis vaccenic and oleic acid. In the presence of another insulin secretagogue, L-leucine, trans vaccenic but not elaidic acid caused a higher response than their cis isomeric fatty acids. The higher potency of trans fatty acids compared with the cis forms was confirmed in perifusion experiments. Both cis and trans C 18:1 fatty acids stimulated insulin secretion in a glucose-dependent manner. Also, glucose oxidation was influenced differentially by the isomers of fatty acids. Glucose oxidation at 16.7 mmol/L glucose was significantly inhibited by oleic and cis vaccenic acid compared with elaidic and trans vaccenic acid, respectively. In summary, our results demonstrate that the fatty acid spatial configuration modulates glucose oxidation and insulin secretion in mouse beta cells.