To better understand the biological significance of Ca(2+), we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca(2+)-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 +/- 2 vs. 7 +/- 1), fewer protein ligands (4 +/- 2 vs. 6 +/- 1), and more water ligands (2 +/- 2 vs. 1 +/- 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H(2)O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H(2)O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 +/- 1 vs. 3 +/- 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix-loop-helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca-O-C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca(2+)-binding proteins.