The transcription factor NF-kappaB is a critical regulator of T cell function that becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-kappaB activation are well understood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-kappaB activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in modulating NF-kappaB activation was examined. NF-kappaB was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-negative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-alpha) activated NF-kappaB normally. Protein kinase C (PKC) theta activation, which is required for NF-kappaB activation, also was defective in these cells. Reexpression of ZAP-70 restored PKCtheta and NF-kappaB activation in response to TCR and CD28 coengagement. p95(vav) (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; however, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wild-type SLP-76 fully restored PKCtheta and NF-kappaB activation in the SLP-76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine phosphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathway in CD3/CD28-stimulated activation of PKC theta and NF-kappaB, and suggest that Vav-1 association with SLP-76 may be important in this pathway.