Calmodulin (CaM) is an essential calcium-binding protein that binds to and activates a diverse population of downstream targets (calmodulin-binding proteins; CaMBPs) that carry out its critical signalling functions. In spite of the central importance of CaM in Ca(2+)-mediated signal transduction pathways in all eukaryotes, many CaMBPs remain to be identified and characterized. SDS-PAGE followed by gel overlay with recombinant, metabolically radiolabelled CaM (Calmodulin-binding Overlay Technique, CaMBOT) is a valuable method for following behavioural, developmental, forensic and physiological changes in total CaMBP populations and to identify candidate CaMBPs for further study. CaMBOT has also been adapted to isolate cDNAs encoding novel CaMBPs in various organisms. Recently, the method was used to examine the CaMBP complement encoded by the Arabidopsis genome and to identify a new family of transcription activators. To add to its diversity, CaMBOT may be useful for finding target proteins for work on phytoremediation and for the screening of pharmaceuticals and toxic agents that, directly or indirectly, affect CaM and its target proteins. This review discusses all of these topics and the role of CaMBOT in characterizing a functional unit of the proteome-proteins regulated by calmodulin.