T-Coffee, for Tree-based consistency objective function for alignment evaluation, is a versatile multiple sequence alignment (MSA) method suitable for aligning virtually any type of biological sequences. T-Coffee provides more than a simple sequence aligner; rather it is a framework in which alternative alignment methods and/or extra information (i.e., structural, evolutionary, or experimental information) can be combined to reach more accurate and more meaningful MSAs. T-Coffee can be used either by running input data via the Web server ( http://tcoffee.crg.cat/apps/tcoffee/index.html ) or by downloading the T-Coffee package. Here, we present how the package can be used in its command line mode to carry out the most common tasks and multiply align proteins, DNA, and RNA sequences. This chapter particularly emphasizes on the description of T-Coffee special flavors also called "modes," designed to address particular biological problems.