AbstractSummary: Molecular dynamics and Monte Carlo, usually conducted in canonical ensemble, deliver a plethora of biomolecular conformations. Proper analysis of the simulation data is a crucial part of biophysical and bioinformatics studies. Sequence alignment problem can be also formulated in terms of Boltzmann distribution. Therefore tools for efficient analysis of canonical ensemble data become extremely valuable. T-Pile package, presented here provides a user-friendly implementation of most important algorithms such as multihistogram analysis and reweighting technique. The package can be used in studies of virtually any system governed by Boltzmann distribution.Availability: T-Pile can be downloaded from: http://biocomp.chem.uw.edu.pl/services/tpile. These pages provide a comprehensive tutorial and documentation with illustrative examples of applications.Contact: dgront@chem.uw.edu.plSupplementary information: Supplementary data are available at Bioinformatics online.