Abstract Motivation: The prediction and annotation of the genomic regions involved in gene expression has been largely explored. Most of the energy has been devoted to the development of approaches that detect transcription start sites, leaving the identification of regulatory regions and their functional transcription factor binding sites (TFBSs) largely unexplored and with important quantitative and qualitative methodological gaps. Results: We have developed ReLA (for REgulatory region Local Alignment tool), a unique tool optimized with the Smith–Waterman algorithm that allows local searches of conserved TFBS clusters and the detection of regulatory regions proximal to genes and enhancer regions. ReLA's performance shows specificities of 81 and 50% when tested on experimentally validated proximal regulatory regions and enhancers, respectively. Availability: The source code of ReLA's is freely available and can be remotely used through our web server under http://www.bsc.es/cg/rela. Contact: david.torrents@bsc.es Supplementary information: Supplementary data are available at Bioinformatics online.