T lymphocytes isolated from human peripheral blood express β1 (VLA) and LFA-1 integrins, but strong binding to integrin ligands occurs only after the delivery of an activation stimulus to the T cell. To gain further insight into activation-dependent regulation of integrin function, we have analyzed integrin activity on three different T-leukemic cell lines: Jurkat, CEM, and H9. This analysis shows important mechanistic differences in integrin regulation. First, phorbol ester treatment results in increased β1 integrin- dependent adhesion of both Jurkat and CEM cells to fibronectin, but decreased adhesion of H9 cells. Second, certain activation stimuli that upregulate β1 integrin activity in peripheral T cells are nonfunctional in these T-cell lines. Third, analysis of a panel of Jurkat mutants lacking surface expression of CD2 and/or CD3 shows that CD2-mediated upregulation of β1 integrin activity is dependent on expression of CD3, whereas CD28-mediated upregulation is not dependent on either CD2 or CD3 expression. Fourth, all T- cell lines tested show an inability to adhere to purified ICAM-1 via LFA-1. The selective alterations in integrin regulation in these cell lines relative to peripheral blood T cells provide important insights into the intracellular processes involved in integrin activation.