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Biologically significant sites in a protein may be identified by contrasting the rates of synonymous (Ks) and non-synonymous (Ka) substitutions. This enables the inference of site-specific positive Darwinian selection and purifying selection. We present here Selecton version 2.2 (http://selecton. bioinfo.tau.ac.il), a web server which automatically calculates the ratio between Ka and Ks (u) at each site of the protein. This ratio is graphically displayed on each site using a color-coding scheme, indicating either positive selection, purifying selection or lack of selection. Selecton implements an assembly of different evolutionary models, which allow for statistical testing of the hypothesis that a protein has undergone positive selection. Specifically, the recently developed mechanisticempirical model is introduced, which takes into account the physicochemical properties of amino acids. Advanced options were introduced to allow maximal fine tuning of the server to the user’s specific needs, including calculation of statistical support of the u values, an advanced graphic display of the protein’s 3-dimensional structure, use of different genetic codes and inputting of a pre-built phylogenetic tree. Selecton version 2.2 is an effective, user-friendly and freely available web server which implements up-to-date methods for computing site-specific selection forces, and the visualization of these forces on the protein’s sequence and structure. INTRODUCTION Current protein sequences have been shaped by a prolonged and extensive evolutionary process. Thus, studying a protein’s evolutionary history may contribute to the inference of the proteins’ properties. Evolutionary conserved sites may be indicative of active sites or of protein–protein interaction domains, while highly variable sites may represent sites subjected to positive Darwinian selection (1,2). Such positively selected sites may be interpreted as being a consequence of molecular adaptation, which confers an evolutionary advantage to the organism. Detecting the level of selection operating on a given protein is enabled by computing o, the ratio between non-synonymous (Ka) and synonymous (Ks) substitutions. Sites showing o values significantly higher than one are indicative of positive Darwinian selection, while sites showing o values significantly lower than one are indicative of purifying selection (2,3). Selecton was first introduced in 2005 (4), and implemented an evolutionary codon model (5) which enabled calculating o at each codon site using a maximumlikelihood (ML) approach. With the advent of sequenced genomes, the comparison of DNA sequences in order to infer meaningful information has become a basic procedure for many researchers. The study of the selection forces operating on a protein, and specifically the attempt to identify positive selection in proteins has been on the rise, and with this rise the number of users of Selecton has increased dramatically. This has motivated us to upgrade the server to include most recent stateof-the-art methods for calculating positive selection. We have thus implemented five evolutionary models (6–8) that enable studying the selection forces operating The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors *To whom correspondence should be addressed. Tel: 972-3-640-7693; Fax: 972-3-642-2046; Email: [email protected]