The underlying mechanisms of glucose-induced time-dependent potentiation in the pancreatic β-cell are unknown. It had been widely accepted that extracellular Ca2+ is essential for this process. However, we consistently observed glucose-induced priming under stringent Ca2+-free conditions, provided that the experiment was conducted in a HEPES-buffered medium as opposed to the bicarbonate (HCO3-)-buffered medium used in previous studies. The critical difference between these two buffering systems is that islets maintain a lower intracellular pH in the presence of HEPES. The addition of HEPES to a HCO3--buffered medium produced a dramatic decrease in the intracellular pH. If it is the lower intracellular pH in islets in a HEPES-buffered medium that is permissive for glucose-induced time-dependent potentiation (TDP), then experimental lowering of intracellular pH by other means should allow TDP to occur in a Ca2+-free HCO3--buffered medium, where TDP normally does not occur. As expected, experimental acidification produced by dimethyl amiloride (DMA) allowed glucose to induce TDP in a Ca2+-free HCO3--buffered medium. DMA also enhanced the priming normally present in HEPES-buffered media. Priming was also enhanced by transient acidification caused by acetate. Experimental alkalinization inhibited the development of priming. In the presence of Ca2+, the magnitude of glucose-induced TDP was higher in a HEPES-buffered medium than in an HCO3--buffered medium. In summary, glucose-induced priming was consistently observed under conditions of low intracellular pH and was inhibited with increasing intracellular pH, irrespective of the presence of extracellular Ca2+. These data indicate that glucose-induced TDP is critically dependent on intracellular pH.