Lymphocyte chemoattractant factor (LCF) is a polypeptide cytokine which induces both cell motility and activation of T lymphocytes. These LCF-induced events demonstrate an absolute requirement for the cell surface expression of CD4. Because many CD4-mediated T lymphocyte activation events have been demonstrated to require the association of the src-related tyrosine kinase p56lck with the cytoplasmic domain of CD4, we examined the role of p56lck in LCF-induced lymphocyte migration in a murine T cell hybridoma line expressing transfected human CD4. LCF induces the catalytic activity of CD4 associated p56lck at chemoattractant concentrations of cytokine. Hybridoma cells that express CD4 with cytoplasmic point mutations which uncouple the CD4-lck association lack both lck enzymatic activity and chemotactic responses to LCF. The enzymatic activity of lck however does not appear to be required for CD4-mediated migratory signal. First, the protein tyrosine kinase inhibitor herbimycin A blocked LCF-induced p56lck activation but had no effect on the LCF-induced motile response. Second, T cell hybridomas expressing a chimeric receptor combining the extracellular domain of human CD4 and murine p56lck which lacked the kinase domain had a normal LCF-induced motile response. We conclude from these observations that CD4-lck coupling is essential for LCF-induced T lymphocyte migration but the motile response is independent of the enzymatic activity of CD4-associated p56lck.