Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH(i)) changes. This study examined the effect of propionate on the pH(i) of bovine neutrophils; specifically if pH(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na+/H+ exchanger and H+ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.