Ca(2+) signaling pathways control many physiological processes in almost all types of animal cells such as fertilization, muscle contraction, hormone release, and learning and memory. Each animal cell type expresses a unique group of molecules from the Ca(2+) signaling 'toolkit' to control spatiotemporal patterns of Ca(2+) signaling. It is generally believed that the complex Ca(2+) signaling 'toolkit' has arisen from the ancestral multicellular organisms to fit unique physiological roles of specialized cell types. Here, we demonstrate for the first time the presence of an extensive Ca(2+) signaling 'toolkit' in the unicellular choanoflagellate Monosiga brevicollis. Choanoflagellates possess homologues of various types of animal plasma membrane Ca(2+) channels including the store-operated channel, ligand-operated channels, voltage-operated channels, second messenger-operated channels, and 5 out of 6 animal transient receptor potential channel families. Choanoflagellates also contain homologues of inositol 1,4,5-trisphosphate receptors. Furthermore, choanoflagellates master a complete set of Ca(2+) removal systems including plasma membrane and sarco/endoplasmic reticulum Ca(2+) ATPases and homologues of 3 animal cation/Ca(2+) exchanger families. Therefore, a complex Ca(2+) signaling 'toolkit' might have evolved before the emergence of multicellular animals.