TR2(L) is a 56-amino-acid polypeptide that has been shown to block TNF cytotoxicity. FE65-like (FE65L) proteins possess this conserved TR2(L) sequence at their C-termini, whereas variations in the sequences are found in the FE65 proteins. To further analyze the antiapoptotic function of TR2(L), here we utilized an isolated murine partial FE65L cDNA that encodes an N-terminal phosphotyrosine-binding: domain (PTB) and the conserved C-terminal TR2(L) sequence. When L929 cells were stably transfected with the FE65L cDNA or its 3' end TR2(L) DNA sequence, these cells became resistant to TNF killing. Replacement of the N-terminal PTB domain with GFP failed to abolish the FE65L-mediated TNF resistance. Ablation of the C-terminal TR2(L) sequence through frame-shift mutation resulted in a complete loss of the FE65L function against TNF. Various protein kinase inhibitors, including lavendustin A, tyrphostin, H7, and staurosporine, which may affect the PTB domain function, could not abolish the FE65L-mediated TNF resistance. A prolonged exposure of L929 cells to these inhibitors for 24 h resulted in cell death, whereas FE65L significantly blocked the cell death. Polyclonal antibodies were generated against a synthetic peptide and shown to interact with a 38-kDa FE65L in L929 cells. Hyaluronidase downregulates the expression of FE65L gene and protein in L929 cells, and this correlates with its enhancement of TNF killing of these cells. Together, our data indicate that the TR2(L) amino acid sequence is an apoptosis-inhibitory domain commonly present in the FE65 and FE65-like family proteins.