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, tumor growth factor (TGF)α showed the highest HK pro-motility activity, reaching ∼80% of the activity in HS. The order of potency was: TGFα>insulin>EGF>heparin binding (HB)-EGF>IGF-1>basic fibroblast growth factor >IL-8>HGF>IL-1>KGF>TGFβ. Interestingly, the combination of TGFα and insulin could duplicate the HK pro-motility activity in HS, although only the TGFα, but not insulin, levels increase in serum over plasma. Addition of neutralizing antibodies against TGFα to serum or depletion of TGFα from serum by immunoprecipitation significantly abolished its HK pro-motility activity. Plasma with added TGFα stimulated HK migration that reached more than 80% of the serum stimulation. Since insulin levels are identical between plasma and serum, we propose that TGFα is the physiologic HK pro-motility factor in HS.