AbstractThe EF‐hand protein with a helix–loop–helix Ca2+ binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca2+ in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF‐hand and EF‐like Ca2+‐binding proteins. The canonical EF‐hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N‐ and C‐terminal EF‐hands within S100 and S100‐like proteins, a new signature profile has been established to allow for the identification of pseudo EF‐hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF‐hands. Furthermore, using the developed patterns, we have identified zero pseudo EF‐hand motif and 467 canonical EF‐hand Ca2+ binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF‐hand motifs are phylogenetically younger than canonical EF‐hand motifs. Our prediction of Ca2+ binding motifs provides not only an insight into the role of Ca2+ and Ca2+‐binding proteins in bacterial systems, but also a way to explore and define the role of Ca2+ in other biological systems (calciomics). Proteins 2006. © 2006 Wiley‐Liss, Inc.