The transcription factor NF-κB plays key roles in inflammatory and immune responses. In different contexts, NF-κB regulates different sets of genes. Previous work had suggested that the methyltransferase Set9 influenced the expression of NF-κB–responsive genes. Ea and Baltimore show that treatment of human embryonic kidney (HEK) 293 cells with a nonspecific methyltransferase inhibitor decreased activation of an NF-κB reporter gene in response to tumor necrosis factor–α (TNF-α). When recombinant Set9 was incubated in vitro with peptides from the p65 subunit of NF-κB, peptides containing Lys37 were methylated. To investigate the function of NF-κB Lys37 methylation in vivo, they developed an antibody specific for this modified form of NF-κB and verified its specificity with a mutant (K37Q) that could not be methylated. In Western blot experiments, the antibody detected methylated NF-κB in the nuclear fractions, but not the cytosol, of cells treated with TNF-α or interleukin-1β (IL-1β). Lys37 methylation in response to TNF-α or IL-1β was undetectable in cells in which Set9 was knocked down with short-interfering RNAs. Selected induction of specific NF-κB–responsive genes was inhibited in cells in which Set9 was knocked down: Induction of the genes encoding TNF-α and the chemokine CXCL10 was decreased, but induction of the gene encoding IκBα was not. Electrophoretic mobility shift assays performed under high salt conditions on nuclear extracts from control or Set9-knockdown cells suggested that Lys37 methylation stabilized a subset of p65-DNA interactions, a finding confirmed by chromatin immunoprecipitation experiments. Induction of the genes encoding TNF-α and CXCL10 was only fully restored by reconstitution of p65-deficient mouse embryo fibroblasts with wild-type p65 and not the K37Q mutant. The authors suggest that this modification may be context-specific and speculate that lysine methylation of this N-terminal lysine (Lys37) may be important for gene activation, whereas lysine methylation of more C-terminal residues (Lys315, Lys316) may be important for termination of activity, a phenomenon that had been reported previously.