The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx-Lbx and NK3-NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila.T he Drosophila melanogaster 93D͞E or NK gene cluster contains six homeobox genes: tinman (tin, NK4), bagpipe (bap, NK3), ladybird late (lbl), ladybird early (lbe), C15 (93Bal), and slouch (slou, NK1) in a gene cluster spanning just 180 kb (1, 2). All six genes possess homeobox sequences of the ANTP class (3), forming a distinct clade within this class along with several NK-related genes (4). The similarity of the genes and their clustered arrangement indicates that they arose through a series of tandem gene duplications, in an analogous way to the Hox gene cluster. We deduce that the NK homeobox gene cluster is ancient, dating at least to the base of the Bilateria. The reasoning is based on the phylogenetic distribution of these genes. Animals from very divergent evolutionary lineages have orthologues of each Drosophila NK gene (except for lbl and lbe, which are recent tandem duplicates). For example, human NKX3.1 and NKX3.2 (BAPX1) genes are orthologues of Drosophila bap, LBX1 and LBX2 genes are orthologues of the ancestral ladybird gene, two human genes equivalent to mouse Nkx1.1 and Nkx1.2 are orthologues of slou, and TLX1, TLX2, and TLX3 (the HOX11 family) are orthologues of C15 (4). It is also likely that human NKX2. 3, NKX2.5, and NKX2.6 are orthologues of Drosophila tin, although sequence similarity is less clear (4, 5). Existence of these human genes implies that the tandem duplications that produced these various NK-related genes had occurred before the divergence of humans and Drosophila and, by implication, before the separation of the Deuterostomia and Ecdysozoa. Therefore, Drosophila NK (and related) genes have remained a tight gene cluster since the origin of these genes Ͼ500 million years (Myr) ago, which implies the existence of a selective reason for the gene clustering in Drosophila.