Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC-MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins.