Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.