Transcriptional regulation is coupled with numerous intracellular signaling processes often mediated by second messengers. Now, growing evidence points to the importance of Ca²⁺, one of the most versatile second messengers, in activating or inhibiting gene transcription through actions frequently mediated by members of the EF‐hand superfamily of Ca²⁺‐binding proteins. Calmodulin and calcineurin, representative members of this EF‐hand superfamily, indirectly regulate transcription through phosphorylation/dephosphorylation of transcription factors in response to a Ca²⁺ increase in the cell. Recently, a novel EF‐hand Ca²⁺‐binding protein called DREAM has been found to interact with regulatory sequences of DNA, thereby acting as a direct regulator of transcription. Finally, S100B, a dimeric EF‐hand Ca²⁺‐binding protein, interacts with the tumor suppressor p53 and controls its transcriptional activity. In light of the structural studies reported to date, this review provides an overview of the structural basis of EF‐hand Ca²⁺‐binding proteins linked with transcriptional regulation. BioEssays 24:625–636, 2002. © 2002 Wiley Periodicals, Inc.