Bio3d is an R package for the comparative analysis of protein structures. It provides tools for processing, organizing, and exploring structure and sequence data. The package includes functions for reading and writing structure, sequence, and dynamic trajectory data, performing atom summaries, selection, re-orientation, superposition, rigid core identification, clustering, distance matrix analysis, structure and sequence conservation analysis, and principal component analysis (PCA). Bio3d takes advantage of the extensive graphical and statistical capabilities of the R environment. The bio3d package employs refined structural superposition and PCA to examine the relationship between different conformers. It uses an iterated superposition procedure to exclude residues with the largest positional differences until only the invariant 'core' residues remain. PCA is then used to examine the relationship between different conformers/structures based on their equivalent residues. The resulting principal components describe the axes of maximal variance of the distribution of structures. Projection of the distribution onto the subspace defined by the largest principal components results in a lower dimensional representation of the structural dataset, termed 'conformer plots'. These plots succinctly display the relationships between different conformers, highlight the major differences between structures, and enable the interpretation and characterization of multiple interconformer relationships. The bio3d comparative analysis results are in good agreement with descriptions established by human experts. The tools provide quantitative and visual information allowing for a more complete appreciation of interconformer relationships. Access to the open source software, full documentation, quick start guide, and example data are available at http://mccammon.ucsd.edu/~bgrant/bio3d/. The structure comparison procedures described here should facilitate the examination of diverse protein families, helping to identify common structural and dynamic features. Such analysis of structural homologues can provide invaluable conformational landmarks useful for assessing both new crystallographic structures and the results of theoretical methods. The current analysis methods may prove valuable to any study where knowledge of backbone flexibility must be modeled. Theoretical studies combined with comparative analysis of structural homologues are an initial step in deciphering possible networks of communication within proteins and understanding allosteric mechanisms.Bio3d is an R package for the comparative analysis of protein structures. It provides tools for processing, organizing, and exploring structure and sequence data. The package includes functions for reading and writing structure, sequence, and dynamic trajectory data, performing atom summaries, selection, re-orientation, superposition, rigid core identification, clustering, distance matrix analysis, structure and sequence conservation analysis, and principal component analysis (PCA). Bio3d takes advantage of the extensive graphical and statistical capabilities of the R environment. The bio3d package employs refined structural superposition and PCA to examine the relationship between different conformers. It uses an iterated superposition procedure to exclude residues with the largest positional differences until only the invariant 'core' residues remain. PCA is then used to examine the relationship between different conformers/structures based on their equivalent residues. The resulting principal components describe the axes of maximal variance of the distribution of structures. Projection of the distribution onto the subspace defined by the largest principal components results in a lower dimensional representation of the structural dataset, termed 'conformer plots'. These plots succinctly display the relationships between different conformers, highlight the major differences between structures, and enable the interpretation and characterization of multiple interconformer relationships. The bio3d comparative analysis results are in good agreement with descriptions established by human experts. The tools provide quantitative and visual information allowing for a more complete appreciation of interconformer relationships. Access to the open source software, full documentation, quick start guide, and example data are available at http://mccammon.ucsd.edu/~bgrant/bio3d/. The structure comparison procedures described here should facilitate the examination of diverse protein families, helping to identify common structural and dynamic features. Such analysis of structural homologues can provide invaluable conformational landmarks useful for assessing both new crystallographic structures and the results of theoretical methods. The current analysis methods may prove valuable to any study where knowledge of backbone flexibility must be modeled. Theoretical studies combined with comparative analysis of structural homologues are an initial step in deciphering possible networks of communication within proteins and understanding allosteric mechanisms.