T-Coffee is a versatile Multiple Sequence Alignment method suitable for aligning most types of biological sequences. The series of protocols presented here show how the package can be used to multiply align proteins, DNA and RNA sequences. The protein section presents controlled cases for PSI-Coffee the homology extended mode suitable for remote homologues, Expresso the structure based multiple aligner and M-Coffee, a meta-version able to combine several third party aligners into one. We then show how the T-RMSD option can be used to produce a functionally informative structure based clustering. RNA alignment procedures are shown for R-Coffee a mode that produces secondary structure based MSAs. DNA alignments are illustrated with Pro-Coffee, a multiple aligner specific of promoter regions. The last sections presents the many reformatting utilities bundled with T-Coffee. The package is an open-source freeware available from www.tcoffee.org.