Hox genes encode transcription factors involved in the specification of segment identity in the early metazoan embryo. These genes are usually clustered and arranged in the same order as they are expressed along the anteroposterior body axis. This conserved genomic organization has suggested the existence of functional constraints acting on the genome organization. Partial disassembly of the Hox gene complex (HOM-C) in Caenorhabditis elegans and in two different Drosophila lineages, however, calls into question whether this cluster organization is absolutely required for proper function. Here we report a new split of the HOM-C discovered in the species of the Drosophila repleta group, which relocated the most anterior gene of the complex, lab, to a distant chromosomal site near the two most posterior Hox genes, abd-A and Abd-B. To investigate the evolutionary consequences of natural rearrangements of the Hox gene complex, the gene lab has been cloned and sequenced in D. buzzatii, a member of the D. repleta group with the split, and in D. virilis, a member of a different species group without the split. The results show that the structure of lab in D. buzzatii is intact and place the breakpoint at least 8 kb from its transcription start site. The nucleotide sequence evolution of lab in the genus Drosophila has been investigated by means of maximum likelihood methods. No significant variation has been observed among lineages in the rate of nucleotide substitution or in the nonsynonymous/synonymous substitution ratio. Seemingly, the relocation of lab has not induced a change in evolution rate or degree of functional constraint. Nevertheless, further work is needed to ascertain whether the lab-pb split has had any effects on gene expression.