Recently, it has been shown that a significant number of evolutionarily conserved human-Fugu noncoding elements function as tissue-specific transcriptional enhancers in vivo, suggesting that distant comparisons are capable of identifying a particular class of regulatory elements. We therefore hypothesized that by juxtaposing human/Fugu and human/mouse conservation patterns we can define conservation criteria for discovering transcriptional regulatory elements specific to mammals. Genome-scale comparisons of noncoding human/Fugu evolutionary conserved elements (ECRs) and their humans/mouse counterparts revealed a particular signature common to human/mouse ECRs (>or=350 bp long, >or=77% identity) that are also conserved in fishes. This newly defined threshold identifies 90% of all human/Fugu noncoding ECRs without the assistance of human-Fugu genome alignments and provides a very efficient filter for identifying functional human/mouse ECRs.