Aldehydes and ketones with one double bond conjugated to the carbonyl group inhibited the enzyme glucose 6-phosphatase, which is embedded in the microsomal membrane. The Michaelis constant, Km and the maximal rate of reaction, V, were affected in a way dependent on the inhibitor's chain-length: trans-2-pentenal and 1-penten-3-one increased Km linearly with concentration and had almost no effect on V, whereas trans-2-nonenal caused a large increase in V but only a small and non-linear change in Km. The effect of the short-chain aldehydes on the kinetic parameters increased with chain-length, but pentenone increased Km more than did trans-2-heptenal and conjugated dienals did not act as inhibitors. Therefore, sterical effects apparently are of importance. Washing the microsomes after incubation with hexenal or heptenal did not substantially decrease the inhibition, but with nonenal the inhibition was reduced by washing. Inhibition by the SH-group blocking reagent p-hydroxymercuribenzoate was competitive to inhibition by the alkenals. It is concluded that the alpha-beta unsaturated oxo-compounds inhibit glucose 6-phosphatase by binding covalently to an important mercapto group and that perturbation of the enzyme's membrane environment also plays a part in the inhibition.