Magnesium ions (Mg(2+)) play a key role in regulating hepatic cellular functions and enzymatic activities. In the present study, we report a concentration-dependent effect of cytosolic Mg(2+) on G6P and pyrophosphate (PPi) transport and hydrolysis in digitonin-permeabilized rat hepatocytes. The stimulatory effect of Mg(2+) on G6P is specific but biphasic, with a maximal effect at a concentration of 0.25 mM, whereas the effect on PPi increases in a dose-dependent manner. Both effects can be abolished by addition of EDTA to the system. Addition of taurocholate, histone-2A, alamethicin or A23187 to the incubation system results in a marked decrease in the Mg(2+) concentration present within the endoplasmic reticulum lumen. Under these conditions, the stimulatory effect of extra-reticular Mg(2+) on G6P transport and hydrolysis is abolished. Taken together, these data suggest that cytosolic Mg(2+) stimulates G6P transport by acting at the level of the substrate binding site of the G6Pase enzymatic complex or the surrounding phospholipid environment. The effect, which is lost when G6P has readily access to the ER lumen, requires physiological endoplasmic reticulum Mg(2+) content.