Vertebrate Hox genes are activated following a temporal sequence that reflects their linear order in the clusters. We introduced two Hoxd transcription units, labeled with lacZ, to an ectopic 5' position in the HoxD complex. Early expression of the relocated genes was delayed and resembled that of the neighboring Hoxd-13. At later stages, locus-dependent expression in distal limbs and the genital eminence was observed, indicating that common regulatory mechanisms are used for several genes. These experiments also illustrated that neighboring genes can share the same cis-acting sequence and that moving genes around in the complex induces novel regulatory interferences. These results suggest that high order regulation controls the activation of Hox genes and highlight three important constraints responsible for the conservation of Hox gene clustering.