Ca2+ carries information pivotal to cell life and death via its interactions with specific binding sites in a protein. We previously developed a novel photoreactive reagent, azido ruthenium (AzRu), which strongly inhibits Ca2+-dependent activities. Here, we synthesized new fluorescent ruthenium-based reagents containing FITC or EITC, FITC-Ru and EITC-Ru. These reagents were purified, characterized and found to specifically interact with and markedly inhibit Ca2+-dependent activities but not the activity of Ca2+-independent reactions. In contrast to many reagents that serve as probes for Ca2+, FITC-Ru and EITC-Ru are the first fluorescent divalent cation analogs to be synthesized and characterized that specifically bind to Ca2+-binding proteins and inhibit their activity. Such reagents will assist in characterizing Ca2+-binding proteins, thereby facilitating better understanding of the function of Ca2+ as a key bio-regulator. (C) 2012 Elsevier Inc. All rights reserved.