The existence and functional activity of a vacuolar-type H⁺-ATPase (vH⁺-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH⁺-ATPase were detectable using RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH⁺-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH⁺-ATPase and Na⁺/H⁺ exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pH_i) was determined in nominally HCO₃^--free, HEPES-buffered NaCl medium containing 20 mM butyrate and after reduction of the extracellular Cl^- concentration ([Cl^-]_e) from 136 to 36 mM. The initial pH_i of REC was 7.4 ± 0.1 in HCO₃^--free, HEPES-buffered NaCl medium and 7.0 ± 0.1 after acid loading with butyrate. Selective inhibition of the vH⁺-ATPase with foliomycin decreased pH_i by 0.19 ± 0.03 pH units. Based on the observed decreases in pH_i resulting from inhibition of vH⁺-ATPase and of subtypes 1 and 3 of NHE, activity of vH⁺-ATPase, NHE3 and NHE1 account for about 30%, 20% and 50% of H⁺-extrusion, respectively. Lowering of [Cl^-]_e induced a pH_i decrease (-0,51 ± 0,03 pH units), impaired pH_i recovery from butyrate-induced acid load, abolished the inhibitory effect of foliomycin and markedly reduced the HOE694- and S3226-sensitive components of pH_i. We conclude that a vH⁺-ATPase is expressed in ovine REC and plays a considerable role in the pH_i regulation of these cells.