The pleiotropic functions of TNF include immune cell activation and inflammation. We previously cloned the cDNA of porcine TNF-Receptor 2 (pTNFR2) to study their contribution to xenograft rejection and found four isoforms generated by two alternative splicings. The processing of ΔE7-10 splicing resulted in loss of the transmembrane region and production of soluble variants, whereas the ΔE4 splicing shortened the TNF-binding domain compromising this function. We hypothesized that the various isoforms display distinct activities and performed structural and expression studies to elucidate their roles. First, we carried out in silico studies to identify key amino acids involved in ligand binding of pTNFR2 that could be affected by exon 4 excision. Next, the relative amount of the two alternative splicings was determined by quantitative RT-PCR in porcine aortic endothelial cells (PAEC) and porcine costal chondrocytes (PCC) in resting and cytokine-stimulated conditions. The predominant isoform was the full receptor, but pro-inflammatory IL-1α and TNFα up-regulated the mRNA expression of all isoforms. For subcellular localization studies, a series of experiments based on immunofluorescence and confocal microscopy were conducted with porcine cells and cells genetically-modified to express the membrane-bound isoforms. Our polyclonal anti-pTNFR2 antibodies recognized both pTNFR2 and pTNFR2ΔE4 by immunostaining. Co-staining with phalloidin in PAEC confirmed that only a small proportion of the receptor was expressed on the cell surface (14% average), whereas recombinant overexpression of pTNFR2 substantially increased the co-localization at the plasma membrane (40%). Transient transfection in Hela cells of pTNFR2-EYFP and pTNFR2ΔE4-EYFP fusion proteins containing the TNF-binding and transmembrane regions produced a very different pattern. A high proportion of the pTNFR2-EYFP co-localized with phalloidin at the plasma membrane (~37%), whereas all pTNFR2ΔE4-EYFP remained inside the cell. In fact, the expression pattern of pTNFR2ΔE4, but not of pTNFR2, was compatible with a predominant expression in the endoplasmic reticulum. Thus, we reveal differences at the molecular and expression levels of pTNFR2 isoforms that provide further insight on the process of xenograft rejection.