TNF is implicated in the attenuation of neutrophil constitutive apoptosis during sepsis. Anti-apoptotic signaling is mediated principally through the TNF receptor-1 (TNFR-1). In adherent neutrophils, when -integrin signaling is activated, TNF phosphorylates TNFR-1, and activates pro-survival and anti-apoptotic signaling. Previously, we identified the -PKC isotype and PI 3-kinase as critical regulators of TNF signaling in adherent neutrophils. Both kinases associate with TNFR-1 in response to TNF and are required for TNFR-1 serine phosphorylation, NFB activation, and inhibition of apoptosis. The purpose of this study was to examine the role of -PKC and PI 3-kinase in assembly of the TNFR-1 signaling complex that regulates NFB activation and anti-apoptotic signaling. Co-immunoprecipitation studies established that PI 3-kinase, -PKC, and TNFR-1 formed a signal complex in response to TNF. -PKC recruitment required both -PKC and PI 3-kinase activity, while PI 3-kinase recruitment was -PKC independent suggesting PI 3-kinase acts upstream of -PKC. An important regulatory step in control of anti-apoptotic signaling is the assembly of the TNFR-1-TRADD-TRAF2-RIP complex that controls NFB activation. Inhibition of either -PKC or PI 3-kinase blocked TNF-mediated recruitment of RIP and TRAF2 to TNFR-1. In contrast, TRADD recruitment was enhanced. Thus, -PKC and PI 3-kinase are positive regulators of TNF mediated association of TRAF2 and RIP with TNFR-1. Conversely, these kinases are negative regulators of TRADD association. These results suggest that -PKC and PI 3-kinase regulate TNF anti-apoptotic signaling at the level of the TNFR-1 receptor through control of assembly of TNFR-1-TRADD-RIP-TRAF2 complex.