The use of biosynthetic fluorescent sensors is an important new approach for imaging Ca²⁺ in cells. Genetically-encoded indicators based on green fluorescent protein, calmodulin, and fluorescence resonance energy transfer (FRET) have been utilized to measure Ca²⁺ in non-mammalian transgenic organisms and provide information about the organization and regulation of Ca²⁺ signaling events in vivo. However, expression of biosynthetic FRET-based Ca²⁺ indicators in transgenic mammals has proven to be problematic. Here, we report transgenic expression of an endoplasmic reticulum (ER) Ca²⁺ biosensor in mouse pancreas. We targeted expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin I promoter. YC3.3er protein expression was limited to pancreatic -cells within islets of Langerhans and absent in the exocrine pancreas and other tissues. Animals developed and matured normally; sensor expression was unaffected by age. Glucose tolerance in transgenic mice was also unaffected, indicating the transgenic biosensor did not impair endocrine pancreas function. ER Ca²⁺ responses following administration of thapsigargin, carbachol, and glucose were measured in individual -cells of intact islets using confocal microscopy, and confirmed the function of the biosensor. We conclude that controlling transgene transcription with a cell-specific promoter permits transgenic expression of FRET-based Ca²⁺ sensors in mammals, and that this approach will facilitate real-time optical imaging of signal transduction events in living tissues.