A fundamental process in limb bud development is the formation of position-dependent cartilage pattern. Cells of the distal mesenchyme maintain positional values as the expression pattern of transcription factors, for example, hox genes, which induce position-related cell differentiation and cell surface differences. Cultured, dissociated limb bud mesenchymal cells segregate from each other, and eventually form cartilage nodules. This sorting out is position-dependent, not cell-type dependent, suggesting that the positional values may be involved. Positional valves were found to be retained in limb bud recombinants. In the chick system, the expression of HoxA13 and HoxD12 was present in the distal half of stage 20 recombinants, whereas these markers were expressed throughout the stages 25 recombinants. In the Xenopus system, multiple digit formation was introduced in limb recombinants, and a position-related relationship between regeneration potency and the multiple digit formation could be established. This determination of multiple digit formation with different stages of limb mesenchyme may be useful in understanding mechanisms of the loss of vertebrate limb regeneration potency.