The mechanism of transfer of magnesium across isolated rumen epithelium was studied in vitro using 28Mg. Isolated pieces of rumen epithelium developed a transmural potential difference of 8.2 +/- 2.3 mV, lumen side negative, during the first hour of incubation when mounted between the two halves of an incubation chamber. A significant net transport of magnesium from lumen to blood was measured when the initial concentration of magnesium in the solutions bathing both surfaces of the epithelium was 2.5 mM. Net flux of magnesium took place against an electrical gradient and was dependent on the presence of the transmural potential difference. The process was saturable at magnesium concentrations of about 5 mM and was markedly reduced by lowering the temperature of the bathing solution, by addition of 10(-4) M ouabain and by extending the time of incubation. The findings strongly support the view that magnesium absorption from the rumen is mediated by an active transport process.