In the present study, we examined the ability of adenosine 3',5'-cyclic monophosphate (cAMP) to reduce elevated levels of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in pancreatic -cells. [Ca²⁺]_i and reduced pyridine nucleotide, NAD(P)H, were measured in rat single -cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca²⁺]_i elevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 µM), and an incretin glucagon-like peptide-1-(7-36) amide (10⁹ M), as well as by glucose (16.7 mM). The [Ca²⁺]_i-reducing effects of cAMP were greater at elevated glucose (8.3-16.7 mM) than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca²⁺]_i-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10-100 nM also reduced sustained [Ca²⁺]_i elevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in -cells. [Ca²⁺]_i-reducing effects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca²⁺ pump. In contrast, [Ca²⁺]_i-reducing effects of cAMP were not altered by ryanodine, an ER Ca²⁺-release inhibitor, Na⁺-free conditions, or diazoxide, an ATP-sensitive K⁺ channel opener. In conclusion, the cAMP-PKA pathway reduces [Ca²⁺]_i elevation by sequestering Ca²⁺ in thapsigargin-sensitive stores. This process does not involve, but is potentiated by, activation of -cell metabolism. Together with the known [Ca²⁺]_i-increasing action of cAMP, our results reveal dual regulation of -cell [Ca²⁺]_i by the cAMP-signaling pathway and by a physiological incretin.