The single amphioxus Hox cluster contains 15 genes and may well resemble the ancestral chordate Hox cluster. We have sequenced the Hox genomic complement of the European amphioxus Branchiostoma lanceolatum and compared it to the American species, Branchiostoma floridae, by phylogenetic footprinting to gain insights into the evolution of Hox gene regulation in chordates. We found that Hox intergenic regions are largely conserved between the two amphioxus species, especially in the case of genes located at the 3' of the cluster, a trend previously observed in vertebrates. We further compared the amphioxus Hox cluster with the human HoxA, HoxB, HoxC, and HoxD clusters, finding several conserved noncoding regions, both in intergenic and intronic regions. This suggests that the regulation of Hox genes is highly conserved across chordates, consistent with the similar Hox expression patterns in vertebrates and amphioxus.