Ca2+, involved in almost all processes of cell life, mediates its activity through reversible interaction with specific binding sites in proteins. Although several Ca2+-dependent activities are known, many of the proteins responsible remain unidentified. Here we describe the synthesis, purification, characterization and potential uses of a new Ca2+-like reagent, azido ruthenium (AzRu), which can be photoactivated. AzRu strongly inhibits Ca2+-dependent activities. AzRu can be used to probe proteins in solution or embedded in membranes. AzRu has no effect on Ca2+-independent or Mg2+-dependent activity. After exposure to ultraviolet irradiation, AzRu binds covalently and specifically to Ca2+-binding proteins, thus providing a new approach for identifying and purifying Ca2+-binding proteins, for characterizing their Ca2+-binding sites and for exploring previously unknown Ca2+-dependent processes. In this protocol we also include a description of the preparation of [103Ru]AzRu, which can be used for labeling Ca2+-binding sites in proteins and identifying previously unknown Ca2+-binding proteins. The preparation of AzRu takes approximately 2–3 days.