Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are high-affinity polypeptide ligands for the EGF receptor, which mediates their biologic activities. In this study, we directly compared the actions of both ligands in promoting keratinocyte motility. We found that normal and tumorigenic human keratinocytes responded to activation of the EGF receptor by either EGF or TGF-alpha; however, the two ligands did not elicit identical responses with regard to cell locomotion. TGF-alpha was more effective than EGF at promoting colony dispersion (cell scattering), in vitro wound closure, and single-cell migration as assessed by phagokinetic track analysis. In contrast, EGF and TGF-alpha evoked identical profiles for DNA synthesis with regard to concentration dependence and magnitude of response in normal keratinocytes and in a squamous cell carcinoma line. The overall pattern of tyrosine phosphorylation of intracellular substrates was similar when cells were stimulated with either growth factor; however, a limited number of differences in the kinetics or magnitude of protein phosphorylation were detected in subcellular fractions. These findings demonstrate that two growth factors implicated in promoting mitogenesis and locomotion may elicit divergent responses with regard to one biologic activity while retaining similar responses for other activities. This suggests that ligand-mediated mitogenic responses may not be tightly coupled to motogenic activity and further illustrates the multifunctional roles of polypeptide growth factors.