This study presents improved cameleons, genetically-encoded fluorescent indicators for calcium (Ca²⁺) that are less sensitive to pH changes. The original cameleons had significant pH interference and uncharacterized Ca²⁺-buffering and cross-reactivity with endogenous CaM signaling pathways. To address these issues, mutations V68L and Q69K were introduced into the acceptor yellow green fluorescent protein, resulting in new cameleons that can measure Ca²⁺ even under significant cytosolic acidification. These improved cameleons show reduced pH sensitivity and are less likely to perturb CaM-dependent signaling pathways. Cameleons consist of a blue or cyan mutant of green fluorescent protein (GFP), calmodulin (CaM), a glycylglycine linker, the CaM-binding domain of myosin light chain kinase (M13), and a green or yellow version of GFP. Ca²⁺ binding to CaM causes intramolecular CaM binding to M13, leading to a conformational change that increases the efficiency of fluorescence resonance energy transfer between the shorter to the longer wavelength mutant GFP. Improved cameleons, such as YC2.1 and YC3.1, have been developed with enhanced resistance to acidification and are more suitable for measuring Ca²⁺ in various cellular environments. The study demonstrates that improved cameleons can measure Ca²⁺ with high accuracy and sensitivity, even in the presence of significant pH changes. They are also less likely to interfere with endogenous CaM signaling pathways. The results show that YC2.1 and YC3.1 have improved Ca²⁺ sensitivity and are suitable for measuring Ca²⁺ in various cellular environments. The study also highlights the importance of considering pH sensitivity when using cameleons for Ca²⁺ measurements. The improved cameleons are also more suitable for long-term measurements and can be used in a variety of cellular environments. The study concludes that the improved cameleons are a valuable tool for measuring Ca²⁺ in various cellular environments.This study presents improved cameleons, genetically-encoded fluorescent indicators for calcium (Ca²⁺) that are less sensitive to pH changes. The original cameleons had significant pH interference and uncharacterized Ca²⁺-buffering and cross-reactivity with endogenous CaM signaling pathways. To address these issues, mutations V68L and Q69K were introduced into the acceptor yellow green fluorescent protein, resulting in new cameleons that can measure Ca²⁺ even under significant cytosolic acidification. These improved cameleons show reduced pH sensitivity and are less likely to perturb CaM-dependent signaling pathways. Cameleons consist of a blue or cyan mutant of green fluorescent protein (GFP), calmodulin (CaM), a glycylglycine linker, the CaM-binding domain of myosin light chain kinase (M13), and a green or yellow version of GFP. Ca²⁺ binding to CaM causes intramolecular CaM binding to M13, leading to a conformational change that increases the efficiency of fluorescence resonance energy transfer between the shorter to the longer wavelength mutant GFP. Improved cameleons, such as YC2.1 and YC3.1, have been developed with enhanced resistance to acidification and are more suitable for measuring Ca²⁺ in various cellular environments. The study demonstrates that improved cameleons can measure Ca²⁺ with high accuracy and sensitivity, even in the presence of significant pH changes. They are also less likely to interfere with endogenous CaM signaling pathways. The results show that YC2.1 and YC3.1 have improved Ca²⁺ sensitivity and are suitable for measuring Ca²⁺ in various cellular environments. The study also highlights the importance of considering pH sensitivity when using cameleons for Ca²⁺ measurements. The improved cameleons are also more suitable for long-term measurements and can be used in a variety of cellular environments. The study concludes that the improved cameleons are a valuable tool for measuring Ca²⁺ in various cellular environments.