Glucose-6-phosphatase (Glc6Pase) is a liver microsomal protein exhibiting a high specificity for glucose-6-phosphate (Glc6P) when present in the intact membrane. On the contrary, Glc6Pase of detergent-treated liver microsomes is able to hydrolyze both Glc6P and mannose-6-phosphate (Man6P) with the same affinities and rates (Km and Vmax) at pH 6.5 [Ajzannay, A., Minassian, C., Riou, J.P., and Mithieux, G. (1993) Eur. J. Biochem. 212, 335-338]. We have carried out competition experiments between constant micromolar concentrations of [U-14C]Glc6P and [U-14C]Man6P and increasing millimolar concentrations of unlabeled Glc6P and Man6P. The rate of hydrolysis of [U-14C]Glc6P was progressively inhibited as the competitor concentration increased, but it was significantly higher in the presence of Man6P than in the presence of Glc6P (twofold higher at 20 mM). In the same manner, the rate of hydrolysis of [U-14C]Man6P was inhibited by increasing concentrations of competitor, but it was significantly higher when the competitor was Man6P than when the competitor was Glc6P. These data provide direct evidence that, in the presence of both Glc6P and Man6P, Glc6Pase is able to exhibit a specific, albeit limited, kinetic behavior towards the former after detergent treatment of the membrane. They also emphasize the role of the membrane in the acquisition of the final specificity of Glc6Pase under physiological conditions.