Previous rotamer libraries showed little significant clustering for asparagine 2 or glutamine 3 values, but none of those studies corrected amide orientations or omitted disordered side chains. The current survey used 240 proteins at <1.7 Å resolution with <50% homology and <30 clashes per thousand atoms (atomic overlap >0.4 Å). All H atoms were added and optimized, and amide orientation was f lipped by 180°if required by H bonding or atomic clashes. A side chain was included only if its amide orientation was clearly determined and if no atom had a B factor >40, alternate conformation, or severe clash; that selection process yielded 1,490 Asn and 863 Gln side chains. Clear clustering was observed for Asn 2 and Gln 3 (except when Gln 2 is trans). For Gln, five major and four minor rotamers cover 87% of examples. For Asn, there are seven backbone-independent rotamers covering 94% of examples plus rotamers specified for strictly ␣-helical, , and left-handed (؉) Asn. Although the strongest inf luence on angles is avoidance of atomic clashes (especially with the NH 2 hydrogens), some Asn or Gln rotamers are inf luenced by favorable van der Waals contacts and others by specific local H-bond patterns.The most important variables for protein conformation are the , , and angles of the backbone, which describe overall tertiary structure. However, the side-chain 1 , 2 , 3 . . . dihedral angles constitute the other half of the conformational specification, determining how the parts of the protein fit together and how the functional groups of the side chains can interact with other molecules. Originally, each -angle distribution was studied separately, but since Ponder and Richards (1), most treatments have been organized around ''rotamers,'' or separate populated clusters in the n-dimensional space of values for a given amino acid type. Libraries of discrete side chain rotamers are widely used for homology modeling, protein redesign, Monte Carlo calculations, and crystallographic electron-density map fitting. The usefulness of the rotamer concept depends on two conditions: (i) the clusters are sharp and separated and (ii) the distributions are not simply independent of one another. Both conditions hold for most side-chain types, but Asn and Gln show notably poor clustering.Asn and Gln 1 and Gln 2 angles are well behaved, with optima near the three staggered values. The major aspect at issue in the analysis of Asn͞Gln conformations is the outermost dihedral angle ( 2 for Asn and 3 for Gln). When the side-chain amide N and O atoms cannot be distinguished, that angle is uncertain by 180°. It involves the rotation of a planar amide relative to a tetrahedral group and has less distinct preferences than a rotation between methyl or methylene groups. The Asn͞Gln amide angles are often omitted from side-chain conformer analyses and are treated differently each time they are included. Different studies have presumed 180°s ymmetry (2-4); have used 0°, ϩ, t, and Ϫ classes (1); have used Ͻ180°and Ͼ180°classes (5); have used bi...