Concentration and voltage dependence of unidirectional magnesium fluxes across the rat descending colon were measured in a modified Ussing-chamber. Mucosa to serosa (ms) magnesium flux exhibits a saturable component, whereas serosa to mucosa (sm) flux is linearly related to the magnesium concentration from 0.125 to 8 mmol/L. At all the concentrations used, ms magnesium transport is higher than the flux in the opposite direction, resulting in net magnesium absorption. Only ms magnesium transport has a voltage-independent, i.e., cellular-mediated component. Magnesium flux from serosa to mucosa, however, is totally voltage-dependent, i.e., purely diffusive and probably constrained to the paracellular pathway. The cellular-mediated fraction of ms magnesium transport is comparable to that found for calcium. Calcium (5 mmol/L) has no influence on ms flux or on the magnesium flux in the opposite direction. Magnesium (5 mmol/L) has no influence on the sm calcium flux but abolishes calcium absorption by decreasing ms calcium flux. The voltage clamp experiments reveal that magnesium has no influence on the cellular-mediated ms calcium transport but exclusively decreases diffusive calcium flux in this direction. Pretreatment with 1 alpha,25-dihydroxyvitamin D3 (250 ng/kg s.c. daily given for 4 days) has no effect on ms magnesium flux but it remarkably stimulates ms calcium transport. These findings suggest that both earth alkaline ions in the descending colon are transported by separate cellular-mediated mechanisms. On the other hand, the similarity of the kinetic parameters as well as the similarity of the voltage-independent ms fluxes would support the assumption that both ions are absorbed by the same mechanism. Hence, the question of whether both ions are absorbed by the same or by distinct transport processes cannot be answered by the present experiments. Diffusive movement across the paracellular route plays an important role in net transport of both ions. Diffusive magnesium flux is equal in both directions. In contrast, for calcium an asymmetry in the paracellular fluxes with a prevalence of the diffusive sm flux over the diffusive fraction of the flux from mucosa to serosa has been reported. The different behavior with respect to the paracellular movement can be explained by the radius of the hydrated magnesium ion, which is smaller than calcium with its hydration shells.