A cDNA sequence coding for the full-length human transforming growth factor alpha (TGF alpha) precursor protein was introduced into and transcribed in cultured human keratinocytes, using a high-titer, high-expression, murine amphotropic retrovirus. Keratinocytes were shown capable of post-translationally modifying the TGF alpha primary translation product, with the subsequent formation of several cell-associated and secreted forms of TGF alpha, at least five of which, including the 50 amino acid mature species, can potentially bind and activate the epidermal growth factor receptor. Cells overexpressing the TGF alpha gene assumed a spindled morphology with long, bipolar filamentous processes and displayed increased locomotion. The soluble, mature form of TGF alpha alone also could induce the observed changes in cell shape and motility when added to keratinocyte cultures exogenously. The effects were dose dependent, and up to fourfold increases in locomotion were caused by TGF alpha in the absence of bovine pituitary extract (BPE). The addition of BPE to high concentrations of TGF alpha further enhanced keratinocyte motility to eightfold over baseline, suggesting a synergistic interaction between the two factors. These experiments demonstrate that keratinocytes can synthesize several forms of TGF alpha and that TGF alpha, besides being mitogenic, may have other important regulatory functions in keratinocytes.