Sonic Hedgehog (SHH) plays a fundamental role in numerous developmental processes including morphogenesis of limbs, nervous system, and teeth. Using a Bayesian alignment algorithm for phylogenetic footprinting we analyzed approximately 28 kb of noncoding DNA in the SHH locus of human and mouse. This showed that the length of conserved noncoding sequences (4196 nt) shared by these species was approximately 3 times larger than the SHH coding sequence (1386 nt). Most segments were located in introns (53%) or within 2-kb regions upstream (16%) or downstream (20%) of the first and last SHH codon. Even though regions more than 2 kb upstream or downstream of the first and last SHH codon represented 57% (16 kb) of the sequence compared, they accounted for only 11% (494 nt) of the total length of conserved noncoding segments. One region of 650 nt downstream of SHH was identified as a putative scaffold/matrix attachment region (SMAR). Human-mouse analysis was complemented by sequencing in apes, monkeys, rodents, and bats, thus further confirming the evolutionary conservation of some segments. Gel-shift assays indicated that conserved segments are targeted by nuclear proteins and showed differences between two cell types that expressed different levels of SHH, namely human endothelial cells and breast cancer cells. The relevance of these findings with respect to regulation of SHH expression during normal and pathologic development is discussed.