Proinflammatory molecules are important for attracting immune effector cells to localized areas of viral infection. One such cellular mechanism facilitating this response is the NF-kappaB transcription factor. While wild-type vaccinia virus expresses multiple products to inhibit NF-kappaB during infection, the attenuated deletion mutant MVA lacks this ability. However, introduction of the wild-type M2L ORF into the MVA genome will re-establish the parental phenotype. As the M2L protein is unique to poxviruses, we characterized it to elucidate its mechanism to quell an inflammatory response. It was discovered that the M2L protein possesses motifs characteristic of ER-localized proteins: an N-terminal signal peptide sequence, C-terminal endoplasmic reticulum (ER) retention and retrieval sequences, and N-linked glycosylation sites. Indeed, the M2L protein was demonstrated to be N-linked glycosylated and expressed early during infection. Furthermore, confocal microscopic analysis revealed that the M2L protein co-localized with cellular ER proteins. Organelle location also affects M2L protein function: the elimination of the N-terminal leader sequence from the M2L protein compromised both its ER location and its ability to inhibit virus-induced NF-kappaB activation. There is only partial ER localization when a second mutant M2L protein lacking potential endoplasmic reticulum retention signal is expressed. However, this C-terminal deleted mutant protein is compromised in its ability to inhibit NF-kappaB activation. Determination of the ER location of the M2L proteins provides important insights for its function in future investigations.