This study presents improvements to cameleons, genetically encoded fluorescent indicators for Ca²⁺ based on green fluorescent protein variants and calmodulin (CaM). The original cameleons suffered from significant pH interference and uncharacterized interactions with endogenous CaM signaling pathways. To address these issues, the researchers introduced mutations V68L and Q69K into the acceptor yellow-green fluorescent protein, reducing its pH sensitivity. This modification allows for Ca²⁺ measurements even under significant cytosolic acidification. The new cameleons, YC2.1 and YC3.1, show a biphasic and monophasic Ca²⁺ dependency, respectively, with improved emission ratios and reduced pH sensitivity. In live cells, YC2.1 and YC3.1 exhibit robust Ca²⁺ responses, with YC2.1 showing a sustained plateau in response to histamine. The study also demonstrates that the fused CaM and M13 domains in cameleons preferentially interact with each other upon Ca²⁺ elevation rather than with separate molecules of CaM or CaM-binding proteins, minimizing interference with endogenous CaM signaling. The improved cameleons are expected to have broader applications in monitoring Ca²⁺ dynamics in various cellular environments.This study presents improvements to cameleons, genetically encoded fluorescent indicators for Ca²⁺ based on green fluorescent protein variants and calmodulin (CaM). The original cameleons suffered from significant pH interference and uncharacterized interactions with endogenous CaM signaling pathways. To address these issues, the researchers introduced mutations V68L and Q69K into the acceptor yellow-green fluorescent protein, reducing its pH sensitivity. This modification allows for Ca²⁺ measurements even under significant cytosolic acidification. The new cameleons, YC2.1 and YC3.1, show a biphasic and monophasic Ca²⁺ dependency, respectively, with improved emission ratios and reduced pH sensitivity. In live cells, YC2.1 and YC3.1 exhibit robust Ca²⁺ responses, with YC2.1 showing a sustained plateau in response to histamine. The study also demonstrates that the fused CaM and M13 domains in cameleons preferentially interact with each other upon Ca²⁺ elevation rather than with separate molecules of CaM or CaM-binding proteins, minimizing interference with endogenous CaM signaling. The improved cameleons are expected to have broader applications in monitoring Ca²⁺ dynamics in various cellular environments.