The authors developed circularly permuted green fluorescent proteins (cpGFPs) to sense calcium (Ca2+) ions in living cells. They engineered a cpGFP by interchanging and reconnecting the amino and carboxyl portions with a short spacer, creating a fluorescent protein named "pericam." Pericam was fused to calmodulin (CaM) and its target peptide, M13, to monitor Ca2+-dependent protein-protein interactions. Three types of pericams were created through mutations near the chromophore: "flash-pericam," which became brighter with Ca2+; "inverse-pericam," which dimmed; and "ratiometric-pericam," which showed a Ca2+-dependent change in excitation wavelength. These pericams were expressed in HeLa cells to monitor free Ca2+ dynamics, such as oscillations in the cytosol and nucleus. High-speed confocal line-scanning microscopy using flash-pericam detected Ca2+ propagation across the nuclear envelope. Ratiometric-pericams were used to simultaneously measure Ca2+ concentrations in the nucleus and mitochondria, revealing that extra-mitochondrial Ca2+ transients caused rapid changes in mitochondrial Ca2+ levels. Split-pericam, created by deleting the linker in flash-pericam, monitored the Ca2+-dependent interaction between CaM and M13. The study demonstrates the potential of cpGFPs as powerful tools for visualizing Ca2+-dependent processes in living cells.The authors developed circularly permuted green fluorescent proteins (cpGFPs) to sense calcium (Ca2+) ions in living cells. They engineered a cpGFP by interchanging and reconnecting the amino and carboxyl portions with a short spacer, creating a fluorescent protein named "pericam." Pericam was fused to calmodulin (CaM) and its target peptide, M13, to monitor Ca2+-dependent protein-protein interactions. Three types of pericams were created through mutations near the chromophore: "flash-pericam," which became brighter with Ca2+; "inverse-pericam," which dimmed; and "ratiometric-pericam," which showed a Ca2+-dependent change in excitation wavelength. These pericams were expressed in HeLa cells to monitor free Ca2+ dynamics, such as oscillations in the cytosol and nucleus. High-speed confocal line-scanning microscopy using flash-pericam detected Ca2+ propagation across the nuclear envelope. Ratiometric-pericams were used to simultaneously measure Ca2+ concentrations in the nucleus and mitochondria, revealing that extra-mitochondrial Ca2+ transients caused rapid changes in mitochondrial Ca2+ levels. Split-pericam, created by deleting the linker in flash-pericam, monitored the Ca2+-dependent interaction between CaM and M13. The study demonstrates the potential of cpGFPs as powerful tools for visualizing Ca2+-dependent processes in living cells.