The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular Ca(2+)-binding proteins, also known as Ca(2+) sensors. A key obstacle to studying many Ca(2+)-sensing proteins is the difficulty in identifying the numerous downstream target interactions that respond to Ca(2+)-induced conformational changes. Among a number of Ca(2+) sensors in the eukaryotic cell, calmodulin (CaM) is the most widespread and the best studied. Employing the mRNA display technique, we have scanned the human proteome for CaM-binding proteins and have identified and characterized a large number of both known and previously uncharacterized proteins that interact with CaM in a Ca(2+)-dependent manner. The interactions of several identified proteins with Ca(2+)/CaM were confirmed by using pull-down assays and coimmunoprecipitation. Many of the CaM-binding proteins identified belong to protein families such as the DEAD/H box proteins, ribosomal proteins, proteasome 26S subunits, and deubiquitinating enzymes, suggesting the possible involvement of Ca(2+)/CaM in different signaling pathways. The selection method described herein could be used to identify the binding partners of other calcium sensors on the proteome-wide scale.