In unwounded skin, human keratinocytes (HKs) are in contact with a plasma filtrate. In an acute wound, HKs come in contact with serum for the first time. Because human serum (HS), but not plasma, promotes HK migration, we speculated that a major HK pro-motility factor in vivo comes from serum. In this study, we compared all of the published growth factors (GFs), reported to promote HK migration, with HS. No single GF could duplicate the HK pro-motility activity in HS. Among these GFs, transforming growth factor-alpha [corrected] showed the highest HK pro-motility activity, reaching approximately 80% of the activity in HS. The order of potency was: TGFalpha > insulin > EGF > heparin binding (HB)-EGF > IGF-1 > basic fibroblast growth factor >IL-8 > HGF > IL-1 > KGF>TGFbeta. Interestingly, the combination of TGFalpha and insulin could duplicate the HK pro-motility activity in HS, although only the TGFalpha, but not insulin, levels increase in serum over plasma. Addition of neutralizing antibodies against TGFalpha to serum or depletion of TGFalpha from serum by immunoprecipitation significantly abolished its HK pro-motility activity. Plasma with added TGFalpha stimulated HK migration that reached more than 80% of the serum stimulation. Since insulin levels are identical between plasma and serum, we propose that TGFalpha is the physiologic HK pro-motility factor in HS.