The intra-islet insulin hypothesis proposes that the decrement in beta-cell insulin secretion during hypoglycemia provides an activation signal for alpha-cells to release glucagon. A more recent hypothesis proposes that zinc atoms suppress glucagon secretion via their ability to open alpha-cell ATP-sensitive K(+) channels. Since insulin binds zinc, and zinc is co-secreted with insulin, we tested whether decreased zinc delivery to the alpha-cell activates glucagon secretion. In streptozotocin-induced diabetic Wistar rats, we observed that switching off intrapancreatic artery insulin infusions in vivo during hypoglycemia greatly improved glucagon secretion (area under the curve [AUC]: control group 240 +/- 261 and experimental group 4,346 +/- 1,259 pg x ml(-1) x 90 min(-1); n = 5, P < 0.02). Switching off pancreatic artery infusions of zinc chloride during hypoglycemia also improved the glucagon response (AUC: control group 817 +/- 107 and experimental group 3,445 +/- 573 pg x ml(-1) x 90 min(-1); n = 6, P < 0.01). However, switching off zinc-free insulin infusions had no effect. Studies of glucose uptake in muscle and liver cell lines verified that the zinc-free insulin was biologically active. We conclude that zinc atoms, not the insulin molecule itself, provide the switch-off signal from the beta-cell to the alpha-cell to initiate glucagon secretion during hypoglycemia.