This study examines the routes by which Mg²⁺ leaves cultured ovine ruminal epithelial cells (REC). Mg²⁺-loaded (6 mM) REC were incubated in completely Mg²⁺-free solutions with varying [Na⁺] and the Mg²⁺ extrusion rate was calculated from the increase of the [Mg²⁺] in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na⁺-salt). In other experiments, REC were also studied for the intracellular free Mg²⁺ concentration (using mag-fura 2), the intracellular Na⁺ concentration (using SBFI), the intracellular cAMP concentration (using an enzyme-linked immunoassay) and Na⁺/Mg²⁺ exchanger existence (using a monoclonal antibody (mab) raised against the porcine erythrocyte Na⁺/Mg²⁺-exchanger). Mg²⁺-loaded REC show a Mg²⁺ efflux which was strictly dependent on extracellular Na⁺. The Mg²⁺ extrusion rate increased from 0.018 ± 0.009 in a Na⁺-free medium to 0.73 ± 0.3 mM/l cells/min in a 145 mM-Na⁺ medium, respectively, and relates to [Na⁺]_e according to a typical saturation kinetic (K_m-value for [Na⁺]_e = 24 mM; V_max = 11 mM/l cells/min). Mg²⁺ efflux was reduced by imipramine (48 %) and increased after application of db-cAMP (55 %) or PGE₂ (17 %). These effects are completely abolished in Na⁺-free media. Furthermore, an elevation of [cAMP]_i led to an [Mg²⁺]_i decrease which amounted to 375 ± 105 µM. The anti-Na⁺/Mg²⁺ exchanger mab inhibits Mg²⁺ extrusion and, moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na⁺/Mg²⁺ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97 %) for Mg²⁺ extrusion and can be assumed to play a considerable role in the process of Mg²⁺ absorption as well as the maintenance of the cellular Mg²⁺ homeodynamics.