A comprehensive database analysis of CH…O hydrogen bonds in 3124 α-helices and their corresponding helix termini has been carried out from a nonredundant data set of high-resolution globular protein structures resolved at better than 2.0 Å in order to investigate their role in the helix, the important protein secondary structural element. The possible occurrence of 5 → 1 CH…O hydrogen bond between the ith residue CH group and (i − 4)th residue CO with C…O ≤ 3.8 Å is studied, considering as potential donors the main-chain Cα and the side-chain carbon atoms Cβ, Cγ, Cδ and Cϵ. Similar analysis has been carried out for 4 → 1 CH…O hydrogen bonds, since the CH…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1. A total of 17,367 (9310 of type 5 → 1 and 8057 of type 4 → 1) CH…O hydrogen bonds are found to satisfy the selected criteria. The average stereochemical parameters for the data set suggest that the observed CH…O hydrogen bonds are attractive interactions. Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 CH…O hydrogen bonds involving methylene Cγ 1 atom as donor in α-helices. This may be an enthalpic compensation for the greater loss of side-chain conformational entropy for β-branched amino acids due to the constraint on side-chain torsion angle, namely, χ1, when they occur in helices. The preference of amino acids for 4 → 1 CH…O hydrogen bonds is found to be more for Asp, Cys, and for aromatic residues Trp, Phe, and His. Interestingly, overall propensity for CH…O hydrogen bonds shows that a majority of the helix favoring residues such as Met, Glu, Arg, Lys, Leu, and Gln, which also have large side-chains, prefer to be involved in such types of weak attractive interactions in helices. The amino acid side-chains that participate in CH…O interactions are found to shield the acceptor carbonyl oxygen atom from the solvent. In addition, CH…O hydrogen bonds are present along with helix stabilizing salt bridges. A novel helix terminating interaction motif, X-Gly with Gly at Ccap position having 5 → 1 CαH…O, and a chain reversal structural motif having 1 → 5 Cα-H…O have been identified and discussed. Our analysis highlights that a multitude of local CH…O hydrogen bonds formed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini. It may be surmised that the main-chain Cα and the side-chain CH that participate in CH…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical NH…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins. Proteins 2004. © 2004 Wiley-Liss, Inc.