ConSurf 2016 is an improved method for estimating and visualizing evolutionary conservation in macromolecules. The ConSurf web server, established over 15 years ago, analyzes the evolutionary patterns of amino and nucleic acids to identify regions important for structure and function. It automatically collects homologues, infers multiple sequence alignments, and reconstructs phylogenetic trees. These data are used in a probabilistic framework to estimate evolutionary rates of each sequence position. New features in ConSurf 2016 include automatic selection of the best evolutionary model, homology modeling of query proteins, prediction of RNA secondary structures, mapping conservation grades onto 2D RNA models, and an advanced view of the phylogenetic tree for interactive rerunning. The server also allows users to select a subtree for refined analysis, improving the detection of functional regions. Visualization improvements include accounting for protein assembly, supporting non-Java-based visualization, and introducing a color-blind friendly palette. ConSurf uses sequence data to estimate evolutionary conservation, with results more informative when viewed on the 3D structure of the macromolecule. The main changes compared to the previous version are summarized in Table 1. ConSurf's method is statistically robust, allowing differentiation between apparent conservation due to short evolutionary time and genuine conservation from purifying selection. It also assigns confidence intervals around calculated evolutionary rates. The superiority of ConSurf over entropy-based methods in predicting protein active sites and identifying biologically active peptides has been previously demonstrated. The new version of ConSurf provides enhanced visualization and analysis capabilities, including the ability to predict RNA secondary structures and homology-based protein structures. It also allows users to refine results using a subset of sequences, improving the detection of functional regions in proteins and nucleic acids.ConSurf 2016 is an improved method for estimating and visualizing evolutionary conservation in macromolecules. The ConSurf web server, established over 15 years ago, analyzes the evolutionary patterns of amino and nucleic acids to identify regions important for structure and function. It automatically collects homologues, infers multiple sequence alignments, and reconstructs phylogenetic trees. These data are used in a probabilistic framework to estimate evolutionary rates of each sequence position. New features in ConSurf 2016 include automatic selection of the best evolutionary model, homology modeling of query proteins, prediction of RNA secondary structures, mapping conservation grades onto 2D RNA models, and an advanced view of the phylogenetic tree for interactive rerunning. The server also allows users to select a subtree for refined analysis, improving the detection of functional regions. Visualization improvements include accounting for protein assembly, supporting non-Java-based visualization, and introducing a color-blind friendly palette. ConSurf uses sequence data to estimate evolutionary conservation, with results more informative when viewed on the 3D structure of the macromolecule. The main changes compared to the previous version are summarized in Table 1. ConSurf's method is statistically robust, allowing differentiation between apparent conservation due to short evolutionary time and genuine conservation from purifying selection. It also assigns confidence intervals around calculated evolutionary rates. The superiority of ConSurf over entropy-based methods in predicting protein active sites and identifying biologically active peptides has been previously demonstrated. The new version of ConSurf provides enhanced visualization and analysis capabilities, including the ability to predict RNA secondary structures and homology-based protein structures. It also allows users to refine results using a subset of sequences, improving the detection of functional regions in proteins and nucleic acids.