max)Sedolisins are acid proteases that are related with the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both tripeptidylpeptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies.We studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows three major clades of which two contain only fungal sequences. One fungal clade contains all known TPPs whereas the other contains the endosedolisins. We identified four cluster specific inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried αhelix that induces a conformational change near the binding pocket. We also identified a total of 13 specificity determining positions (SDPs) that are divided over three SDP sub-networks. The major SDP network contains eight SDPs and modeling of virtual mutants suggests a key role for the W307A or F307A substitution. This is accompanied by a group of four SDPs that physically interact at the interface of the catalytic domain and the enzyme's prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. The additional small SDP networks appear to be linked to the major network all together explaining the hypothesizes functional diversification of fungal sedolisins.