We present the second and improved release of the TOUCAN workbench for cis-regulatory sequence analysis. TOUCAN implements and integrates fast state-of-the-art methods and strategies in gene regulation bioinformatics, including algorithms for comparative genomics and for the detection of cis-regulatory modules. This second release of TOUCAN has become open source and thereby carries the potential to evolve rapidly. The main goal of TOUCAN is to allow a user to come to testable hypotheses regarding the regulation of a gene or of a set of co-regulated genes. TOUCAN can be launched from this location: http://www.esat.kuleuven.ac.be/similar to saerts/software/toucan.php.