Patterning of the vertebrate limb appears to be mediated by position-specific cell-cell interactions. Evidence for these interactions stem from grafting experiments in which posterior (zone of polarizing activity or ZPA) tissue is placed into an anterior site in the developing chick limb. This manipulation results in supernumerary digits, comprised primarily of anterior cells. Thus, posterior (ZPA) cells produce a signal to which anterior cells are responsive. This is an excellent assay for ZPA activity. In an attempt to characterize and understand patterning mechanisms present in the vertebrate limb during development in vitro culture techniques have been coupled with this in vivo assay system. Cultured mouse limb bud cells have been assayed for ZPA activity by grafting into the developing chick limb bud. Because of the transient nature of ZPA signaling, both in vivo and in vitro, patterning mechanisms within the limb have not been elucidated. We have developed a culture technique, termed microdissociation, that seeks to characterize the components necessary for the maintenance of ZPA activity. Using these characteristics we have identified a maintenance factor, basic fibroblast growth factor (bFGF), that can replace all of the ZPA activity observed in microdissociate cultures In vitro conversion from anterior (non-signaling) to ZPA (signaling) has been demonstrated with retinoic acid, suggesting that anterior cells have the potential to become ZPA cells with the right stimulus. We have investigated the positional stability of anterior cells in vitro and in vivo. Cultured anterior cells without prior treatment undergo a conversion to ZPA suggesting that during normal development a repressor exists in this region of the limb. We have implanted paper carriers soaked in trypsin into the anterior of the chick limb in an attempt to remove the putative endogenous repressor. We have found that trypsin treatment is effective in stimulating a supernumerary response, implicating a repressor in the normal patterning of the limb