We examined the effect of reducing ambient and intracellular free Mg ion ([Mg]i) concentrations on insulin action in epididymal adipocytes from male Sprague-Dawley rats in terms of (1) cellular transport of nonmetabolizable 2-deoxyglucose, (2) [U-14C]glucose oxidation to CO2, and (3) D-[3H]glucose incorporation into triglycerides. There were no significant differences in basal or insulin-stimulated transport of 2-deoxyglucose between adipocytes cultured in physiologic (1.24 mmol) or low (0.16 mmol) Mg for up to 24 hours. In contrast, insulin-stimulated but not basal [U-14C]glucose oxidation to CO2 was significantly reduced in adipocytes cultured in low versus physiologic Mg (P < .05 to .01). Similarly, there were no differences in basal glucose incorporation into triglycerides between cells cultured in low or physiologic Mg media for up to 24 hours. However, long-term (24-hour) but not short-term (2-hour) exposure of cells to low Mg was associated with a significant 30% reduction in insulin-stimulated D-[3H]glucose incorporation into triglycerides. When adipocytes incubated in low Mg were reincubated in high Mg (1.24 or 5 mmol) for 30 minutes, normal insulin-stimulated D-[3H]glucose incorporation into triglycerides was restored. Incubation of adipocytes in low Mg (0.16 mmol) for 24 hours resulted in a significant decrease in [Mg]i (264 +/- 89 v 437 +/- 125 micromol/cell [mean +/- SEM]) as compared with cells incubated in physiologic Mg (1.24 mmol; P < .01). These data support a role for intracellular Mg deficiency in the development of insulin resistance and suggest that the effect occurs at a site(s) distal to glucose entry into the cell. The effect of Mg deficiency on insulin action appears to be reversible.