ProtTest is a tool for selecting the best-fit model of protein evolution for a given protein sequence alignment. It is based on the Phyml program for maximum likelihood (ML) optimizations and uses the PAL library for handling protein alignments and trees. ProtTest is available for Mac OSX, Linux, and Windows and can be run via a GUI, command-line, or web. It implements 64 empirical models, including WAG, mtREV, Dayhoff, JTT, VT, Blosum62, CpREV, and RtREV under +F, +G, +I, and their combinations. The program calculates likelihood under each candidate model, estimates model parameters, and allows users to choose a model selection strategy (AIC, AICc, BIC) to rank model fits, estimate model-averaged parameters, or measure parameter importance. The sample size is set to the number of positions in the alignment by default. Other options attempt to account for both the number of sequences and their redundancy. ProtTest also allows restricting the set of candidate models (only in the GUI version) and outputting the tree corresponding to the best model. The program is available under the GNU license from http://darwin.uvigo.es. It was developed by Federico Abascal, Rafael Zardoya, and David Posada. The work was financially supported by a research grant in bioinformatics from the Fundación BBVA (Spain).ProtTest is a tool for selecting the best-fit model of protein evolution for a given protein sequence alignment. It is based on the Phyml program for maximum likelihood (ML) optimizations and uses the PAL library for handling protein alignments and trees. ProtTest is available for Mac OSX, Linux, and Windows and can be run via a GUI, command-line, or web. It implements 64 empirical models, including WAG, mtREV, Dayhoff, JTT, VT, Blosum62, CpREV, and RtREV under +F, +G, +I, and their combinations. The program calculates likelihood under each candidate model, estimates model parameters, and allows users to choose a model selection strategy (AIC, AICc, BIC) to rank model fits, estimate model-averaged parameters, or measure parameter importance. The sample size is set to the number of positions in the alignment by default. Other options attempt to account for both the number of sequences and their redundancy. ProtTest also allows restricting the set of candidate models (only in the GUI version) and outputting the tree corresponding to the best model. The program is available under the GNU license from http://darwin.uvigo.es. It was developed by Federico Abascal, Rafael Zardoya, and David Posada. The work was financially supported by a research grant in bioinformatics from the Fundación BBVA (Spain).