The K⁺-insensitive component of Mg²⁺ influx in primary culture of ruminal epithelial cells (REC) were examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg²⁺ concentration ([Mg²⁺]_i), Mg²⁺ uptake, and intracellular pH (pH_i) were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg²⁺]_i of REC increased from 0.56 ± 0.14 to 0.76 ± 0.06 mM, corresponding to a Mg²⁺ uptake rate of 15 µM/min. Exposure to butyrate did not affect Mg²⁺ uptake (12 µM/min), but it was stimulated (by 84 ± 19 %) in the presence of CO₂/HCO₃^-. In contrast Mg²⁺ uptake was strongly diminished if REC were suspended in a HCO₃^--buffered high-KCl medium (22.3 ± 4 µM/min) rather than in HEPES-buffered high-KCl medium (37.5 ± 6 µM/min). After switching from high-Cl^- to low-Cl^- solution [Mg²⁺]_i was significantly reduced from 0.64 ± 0.09 to 0.32 ± 0.16 mM and the CO₂/HCO₃^--stimulated Mg²⁺ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg²⁺ uptake by 64 and 40 % of control values, respectively. Specific blockers of V-type H⁺-ATPase (bafilomycin A₁ and foliomycin) reduced the [Mg²⁺]_i (by 36 %) and Mg²⁺ influx into REC (by 38 %). We interpret these data to reflect that the K⁺-insensitive Mg²⁺ influx into REC is mediated by a cotransport of Mg²⁺ and Cl^- and is energized by an H⁺-ATPase. The coupling between ruminal fermentation products (CO₂, SCFA) and Mg²⁺ transport is indirect and may result from a modulation of the H⁺-ATPase activity.