Elevated glucose concentrations cause Ca²⁺ influx and the exocytotic release of insulin from pancreatic islet -cells. Whether increases in cytosolic free Ca²⁺ concentration also mobilize Ca²⁺ from intracellular stores ("Ca²⁺-induced Ca²⁺ release") is unresolved. Endoplasmic reticulum-targeted "Cameleons" have previously been used to explore the involvement of ER Ca²⁺ release in these cells, albeit with differing conclusions (Varadi & Rutter, 2002, Diabetes, 51, Supp1, S190-201; Graves & Hinkle, 2003, Endocrinology, 144(8):3565). Cameleons comprise two spectrally-shifted GFPs, enhanced cyan and yellow fluorescent protein (ECFP or EYFP, respectively) whose orientation is affected by Ca²⁺, changing intramolecular fluorescence resonance energy transfer. By measuring pH in the cytosol and ER lumen, we demonstrate that high K⁺ concentrations (> 20 mM) acidify both compartments in clonal MIN6 -cells when external bicarbonate concentrations are low (< 5 mM), interfering with measurements using Ycam-2 and Ycam-4ER. However, when intracellular pH is strongly buffered (24 mM HCO₃^-) glucose or cell depolarisation increase ER [Ca²⁺] monitored with Ycam-4ER. KCl-induced increases in ER [Ca²⁺] were diminished when intracellular stores were sensitized with 1 mM caffeine, and inhibited by pre-treatment with ryanodine. Furthermore, preincubation with ryanodine tended to slow the falling phase of the ER Ca²⁺ transient following cell depolarisation with KCl and reduced the peak cytosolic [Ca²⁺]. By contrast, stimulation with glucose increased ER [Ca²⁺] both in the absence and presence of caffeine or ryanodine. These observations suggest that Ca²⁺-induced ER Ca²⁺ release can occur in -cells under some conditions, but may not be essential for glucose-stimulated insulin secretion.