TNF-α induced apoptosis in a feline fibroblastic cell line (CRFK) infected with FIV but not in its uninfected control. In this study, to understand the molecular basis of the different susceptibilities to TNF-α between FIV-infected and uninfected cells, we examined the expression of TNF receptors and the activation of the caspase and NF-κB pathways. Expression levels of TNFR I and TNFR II mRNAs were similar between uninfected and FIV-infected CRFK cells. To understand the role of caspases in TNF-α-induced apoptosis, we examined the effect of three different classes of caspase inhibitors, Z-VAD-FMK, Ac-YVAD-CMK, and Z-DEVD-FMK, on the TNF-α-induced apoptosis in FIV-infected cells. Pretreatment with each of these caspase inhibitors protected FIV-infected CRFK cells from TNF-α-induced cell death. Moreover, one of the caspase substrates, poly(ADP-ribose) polymerase, was shown to be cleaved after TNF-α treatment in FIV-infected CRFK cells but not in uninfected CRFK cells. Electrophoretic mobility shift assay using an NF-κB motif oligonucleotide and promoter assay using an NF-κB luciferase reporter construct indicated that TNF-α treatment had induced activation of NF-κB in both FIV-infected and uninfected CRFK cells. The present study indicates that TNF-α-induced apoptosis in FIV-infected CRFK cells is mediated by the activation of the caspase cascade, but not by either upregulation of TNF receptor or inhibition of NF-κB.