Clade B serine proteinase inhibitors (serpins) are intracellular proteins, whereas most of their identified targets are extracellular. A proposed intracellular role for these inhibitors is protection from apoptosis. We investigated the contribution of serpinB2 (plasminogen activator inhibitor-2, PAI-2) activity in TNF-alpha-induced apoptosis. PAI-2 is expressed in many normal and transformed cell types, particularly after stimulation with inflammatory cytokines. PAI-2 has been linked to protection from TNF-alpha-induced apoptosis, and a stabilizing interaction with the retinoblastoma protein (Rb1) has been proposed. We examined the activity of PAI-2 in TNF-alpha-induced apoptosis using HeLa, Isreco-1 and HT1080 cell lines. Stimulation with TNF-alpha protected each cell type from apoptosis induced by TNF-alpha and cycloheximide. Protection correlated with an increase in PAI-2 expression in IS-1 and HT1080 cells but not in HeLa cells where PAI-2 mRNA and protein were undetectable. PAI-2 was overexpressed in each cell type but gave no protection from TNF-alpha-induced apoptosis measured by cell viability, annexinV binding and caspase-3/7 activity. We detected wild-type Rb1, unchanged TNF receptor levels and induction of other apoptosis-protective factors in all cell types. In conclusion, elevated PAI-2 levels do not protect cells from TNF-alpha-induced apoptosis, and the protective effect of prior stimulation with TNF-alpha does not require PAI-2.