Accurate and comprehensive information about the nucleotide sequence specificity of trans-acting factors (TFs) is essential for computational and experimental analyses of gene regulatory networks. We present the Yeast Transfactome Database, a repository of sequence specificity models and condition-specific regulatory activities for a large number of DNA- and RNA-binding proteins in Saccharomyces cerevisiae. The sequence specificities in TransfactomeDB, represented as position-specific affinity matrices (PSAMs), are directly estimated from genomewide measurements of TF-binding using our previously published MatrixREDUCE algorithm, which is based on a biophysical model. For each mRNA expression profile in the NCBI Gene Expression Omnibus, we used sequence-based regression analysis to estimate the post-translational regulatory activity of each TF for which a PSAM is available. The trans-factor activity profiles across multiple experiments available in TransfactomeDB allow the user to explore potential regulatory roles of hundreds of TFs in any of thousands of microarray experiments. Our resource is freely available at http://bussemakerlab.org/TransfactomeDB/