Because peripheral blood mononuclear cells play an important role in the perpetuation of the autoimmune process in rheumatoid arthritis (RA) and because the maintenance of these cells might be caused by the dysregulation of apoptosis, we investigated the apoptosis susceptibility of peripheral blood mononuclear cells from patients with RA. Freshly separated peripheral blood lymphocytes were stained for apoptosis markers (CD95, Bax, Bcl-2, TNF receptor) and for an activation marker (CD45-RO), and the apoptosis frequency of cells bearing these markers were assessed by the terminal-deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling method and nuclear condensation analysis with laser scanning cytometry. Also, the ability of CD4(+) and CD8(+) T-cell populations to undergo apoptosis was investigated with 24-hour culture in medium alone or with different apoptosis inducers (anti-CD3, anti-CD95, anti-TNF receptor). Laser scanning cytometry analysis was used to enumerate the phenotype and apoptosis ratios of both freshly isolated and cultured lymphocytes. Quantitative ELISA was performed to detect plasma levels of TNF-alpha and soluble Fas ligand. Furthermore, we studied the relationship between marked apoptotic defects in patients with RA and the severity of clinical disease. CD4(+) T-cell counts in patients with RA were elevated compared with controls. A decreased rate of anti-CD95-mediated apoptosis was found within the CD4(+) and CD8(+) lymphocytic subpopulations. In patients with RA, decreased Bax expression and decreased apoptosis rate within the Bax-positive cells were found, whereas Bcl-2 expression was elevated. The CD45-RO expression was higher, whereas the apoptosis within CD45-RO(+) cells were decreased in RA. Evaluation of plasma soluble Fas ligand revealed significantly decreased levels in patients compared with controls. The reduced susceptibility to CD95-mediated apoptosis may contribute to the expansion of an activated CD4(+) lymphocyte subpopulation and thus to the maintenance of peripheral autoreactive T-cell clones in RA. We also revealed a relationship between in vitro demonstrated lymphocyte apoptosis defects and clinical disease activity.