We have developed mice in which a purposed designed Ca²⁺ indicator is expressed in a lineage-specific fashion for the examination of Ca²⁺ signalling in vivo. The indicator, GCaMP2, is stable at physiological temperatures and displays excellent signal strength, dynamic range, and on/off kinetics in vivo. Lines of mice expressing GCaMP2 in the heart under control of a tetracylcline transactivator have been created. These mice report cellular Ca²⁺ transients over all regions of the heart with every beat. Experiments have characterized the kinetic limitations of GCaMP2 in simultaneous Rhod2 recordings in isolated, perfused hearts from humanely killed animals. Finally, we have used cardiac GCaMP2 mice to examine early embryonic development of the heart. These experiments provide new insight into the development of the SA and AV nodal regions of the heart and demonstrate the utility of this approach for the examination of complex physiological functions in mammals, in vivo.