Interleukins (IL) 1 and 6 are important cytokines that function via the activation, respectively, of the transcription factors NF-κB and STAT3. We have observed that a specific type of κB DNA sequence motif supports both NF-κB p65 homodimer binding and cooperativity with non-tyrosine-phosphorylated STAT3. This activity, in contrast to that mediated by κB DNA motifs that do not efficiently bind p65 homodimers, is shown to be uniquely dependent upon signal transduction through the carboxyl terminus of TRAF6. Furthermore, STAT3 and p65 are shown to physically interact, in vivo, and this interaction appears to inhibit the function of “classical” STAT3 GAS-like binding sites. The distinct p50 form of NF-κB is also shown to interact with STAT3. However, in contrast to p65, p50 cooperates with STAT3 bound to GAS sites. These data argue for a novel transcription factor cross-talk mechanism that may help resolve inconsistencies previously reported regarding the mechanism of IL-1 inhibition of IL-6 activity during the acute-phase response.