HIV-tat protein, like TNF, activates a wide variety of cellular responses, including NF-κB, AP-1, c-Jun N-terminal kinase (JNK), and apoptosis. Whether HIV-tat transduces these signals through the same mechanism as TNF is not known. In the present study we investigated the role of the T cell-specific tyrosine kinase p56 lck in HIV-tat and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, an isogeneic lck-deficient T cell line. Treatment with HIV-tat protein activated NF-κB, degraded IκBα, and induced NF-κB-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56 lck kinase. These effects were specific to HIV-tat, as activation of NF-κB by PMA, LPS, H 2 O 2, and TNF was minimally affected. p56 lck was also found to be required for HIV-tat-induced but not TNF-induced AP-1 activation. Similarly, HIV-tat activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. HIV-tat also induced cytotoxicity, activated caspases, and reactive oxygen intermediates in Jurkat cells, but not in JCaM1 cells. HIV-tat activated p56 lck activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56 lck tyrosine kinase reversed the HIV-tat-induced NF-κB activation and cytotoxicity. Overall, our results demonstrate that p56 lck plays a critical role in the activation of NF-κB, AP-1, JNK, and apoptosis by HIV-tat protein but has minimal or no role in activation of these responses by TNF.