The effects of glucose and acetylcholine (ACh) on the intracellular free Ca2+ ion concentration ([Ca2+]i) were measured using fura-2 microspectrofluorimetry in individual rat pancreatic beta-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The average [Ca2+]i was 139 +/- 2.2 nM (n = 84) in the presence of 4.4 mM glucose. At 17.8 mM, glucose caused transient or sustained increases in [Ca2+]i in some individual beta-cells (15% of the total cells tested). However, the majority of glucose (17.8 mM)-nonresponsive cells responded to ACh, cholecystokinin-8, and K+. ACh at 10(-4) M stimulated increases in [Ca2+]i in most of the glucose-nonresponsive beta-cells in the presence of 4.4 mM glucose, and the effect was concentration dependent. High concentrations of glucose potentiated ACh-induced increases in [Ca2+]i observed in some of the glucose-nonresponsive beta-cells (glucose-sensitive cells), demonstrating that the function of some beta-cells is affected by the interaction of glucose with ACh. However, glucose did not affect the ACh-induced increase in [Ca2+]i in other glucose-nonresponsive cells (glucose-insensitive cells). These data strongly indicate that there are populations of beta-cells that exhibit different [Ca2+]i responses to glucose.