We have developed a web-based query tool, Whole-Genome rVISTA (WGRV), that determines enrichment of transcription factors (TFs) and associated target genes in sets of co-regulated genes. WGRV enables users to query databases containing pre-computed genome coordinates of evolutionarily conserved transcription factor binding sites in the proximal promoters (from 100 bp to 5 kb upstream) of human, mouse and Drosophila genomes. TF binding sites are based on position-weight matrices from the TRANSFAC Professional database. For a given set of co-regulated genes, WGRV returns statistically enriched and evolutionarily conserved binding sites, mapped by the regulatory VISTA (rVISTA) algorithm. Users can then retrieve a list of genes from the query set containing the enriched TF binding sites and their location in the query set promoters. Results are exported in a BED format for rapid visualization in the UCSC genome browser. Flat files of mapped conserved sites and their genomic coordinates are also available for analysis with stand-alone software.