Short chain fatty acids (SCFAs) and CO2 have been shown to stimulate net Mg2+ efflux from the isolated reticulorumen in vivo. To investigate the underlying mechanisms of Mg2+ transport we performed Ussing chamber and microelectrode experiments and measured 28Mg2+ fluxes across sheep rumen epithelium in vitro. In the presence of SCFAs mucosal-to-serosal Mg2+ flux (JMgm-s) amounted to 82·3 ± 7·8 nmol cm-2 h-1 and serosal-to-mucosal Mg2+ flux (JMgs-m) to 3·2 ± 0·7 nmol cm-2 h-1. Replacing SCFAs with gluconate caused a 50 % reduction of JMgm-s, whereas JMgs-m was not affected. Among the SCFAs, n-butyrate was more effective in stimulating JMgm-s than acetate, propionate or iso-butyrate. Eliminating HCO3--CO2 from SCFA-containing solutions did not affect Mg2+ fluxes, whereas the same replacement in SCFA-free solutions led to a further reduction in JMgm-s. JMgm-s decreased after the addition of ethoxyzolamide to SCFA-free, bicarbonate buffered solutions. Decreasing mucosal pH from 6·4 to 5·4 increased JMgm-s in SCFA-free, bicarbonate buffered solutions. SCFAs had no effect on the apical membrane potential of rumen epithelial cells. The experiments show that both SCFAs and CO2 stimulate Mg2+ transport through an increase in JMgm-s, most probably via stimulation of a Mg2+-2H+ exchange mechanism. SCFAs may have additional metabolic effects on Mg2+ transport.