We have investigated the mechanism by which amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit glucose-stimulated medium acidification in the fission yeast Schizosaccharomyces pombe. The addition of glucose to an unbuffered suspension of cells results in the extrusion of acid. This process was inhibited by diethylstilbestrol (DES), an inhibitor of the H(+)-ATPase (IC50 71 microM), and also by amiloride (IC50 824 microM) and EIPA (IC50 203 microM). The presence of 100 mM NaCl reduced the degree of inhibition observed for amiloride and EIPA, but had no effect on inhibition by DES. N-Methylglucosamine partially protected the cells against the effect of amiloride, but choline chloride did not, suggesting that sodium may be important in the action of amiloride. To establish the site of action of amiloride and EIPA, ATP hydrolysis assays were performed on isolated plasma membranes. H(+)-ATPase activity was inhibited by orthovanadate, but not by amiloride or EIPA. However, both amiloride and EIPA were found to inhibit the incorporation of radioactivity from labelled glucose in S. pombe, with IC50 values of 879 and 272 microM for amiloride and EIPA respectively. Again, 100 mM NaCl was found to reduce the effectiveness of inhibition. Amiloride had no effect on the uptake of 2-deoxyglucose under the same conditions, indicating that amiloride does not inhibit the glucose transporter. We propose that amiloride and EIPA disrupt glucose-induced acidification by inhibiting glucose metabolism.