MOTIVATION: Mutating residues into alanine (alanine scanning) is one of the fastest experimental means of probing hypotheses about protein function. Alanine scans can reveal functional hot spots, i.e. residues that alter function upon mutation. In vitro mutagenesis is cumbersome and costly: probing all residues in a protein is typically as impossible as substituting by all non-native amino acids. In contrast, such exhaustive mutagenesis is feasible in silico. RESULTS: Previously, we developed SNAP to predict functional changes due to non-synonymous single nucleotide polymorphisms. Here, we applied SNAP to all experimental mutations in the ASEdb database of alanine scans; we identi.ed 70% of the hot spots (>or=1 kCal/mol change in binding energy); more severe changes were predicted more accurately. Encouraged, we carried out a complete all-against-all in silico mutagenesis for human glucokinase. Many of the residues predicted as functionally important have indeed been con.rmed in the literature, others await experimental veri.cation, and our method is ready to aid in the design of in vitro mutagenesis. AVAILABILITY: ASEdb and glucokinase scores are available at http://www.rostlab.org/services/SNAP. For submissions of large/whole proteins for processing please contact the author.