1. N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new non-sulphonylurea oral hypoglycaemic agent which stimulates insulin release by increasing cytosolic Ca2+ concentration ([Ca2+]i) in beta-cells. 2. We studied comparative effects of A-4166 and sulphonylureas on [Ca2+]i, measured by dual-wavelength fura-2 microfluorometry, in single rat pancreatic beta-cells under normal conditions and conditions where glucose metabolism was inhibited. 3. A glucokinase inhibitor, mannoheptulose (10 mM), a mitochondrial respiratory inhibitor, KCN (100 microM), and uncouplers, dinitrophenol (DNP, 50 microM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.3 microM), were used to abolish glucose-induced increases in [Ca2+]i in a reversible manner. 4. Under control conditions, A-4166 was one order more potent than tolbutamide in increasing [Ca2+]i, and maximal responses were evoked by 30 microM A-4166 and 300 microM tolbutamide. These equipotent concentrations were employed for the comparative study where glucose metabolism was inhibited. 5. In the presence of mannoheptulose, [Ca2+]i responses to tolbutamide, but not those to A-4166, were attenuated in a reversible manner. 6. KCN, DNP and FCCP inhibited [Ca2+]i responses to tolbutamide to a much greater extent than those to A-4166. Responses to tolbutamide even at 3.3 times the equipotent concentration (1000 microM) were also markedly attenuated by these inhibitors. Responses evoked by another sulphonylurea, gliclazide, were inhibited by DNP to a larger extent than A-4166-induced responses. 7. The results indicate that A-4166 acts more effectively than sulphonylureas to increase [Ca2+]i in beta-cells during metabolic inhibition.