The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. When performing anion competition experiments in the washed reticulo-rumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a SCFA mixture (15 or 30 mM of each acetate, propionate and butyrate). Disappearance rates of acetate and propionate, but not butyrate, (initial concentration, 25 mM each) were diminished by 40 mM or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 mM to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently while sulphate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM; but this doubling was almost abolished when HCO₃^- was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K_m = 54 mM) and a bicarbonate-independent component (K_m = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 µM diisothiocyanatostilbene 2,2-disulfonic acid (DIDS), 400 µM p-chloromercuribenzene sulfonic acid (pCMBS), 800 µM p-hydroxymercuribenzoic acid (pHMB) or 100 µM phloretin had no effects on acetate uptake although the latter two inhibited L-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein-coupled but not via monocarboxylate cotransporter (MCT).