Monoclonal antibodies and chemotherapy can be effective, but not curative, therapy for non-Hodgkin s lymphoma (NHL). Rituximab acts by several mechanisms, including directly signaling apoptosis of CD20+ cells via the mitochondrial pathway involving caspase 9. Chemotherapy also activates this apoptotic pathway. TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. Thus, we investigated the effects of agonistic monoclonal antibodies to TRAIL-R1 and -R2 on NHL cell lines (DoHH2, WSU-FSCCL and FC-TxFL2, each t(14;18)+, EBV-). TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) antibodies were from Human Genome Sciences (Rockville, MD). FC-TxFL2 expressed the highest cell surface levels of TRAIL-R1 (DR4, target of HGS-ETR1) and TRAIL-R2 (DR5, target of HGS-ETR2). WSU-FSCCL expressed lower, but significant, levels of each. DoHH2 expressed dim TRAIL-R1 and the lowest levels of TRAIL-R2 of the 3 cell lines. IC50 (MTT assay after 72 hr incubation with antibody) was 0.25mcg/ml for WSU-FSCCL and FC-TxFL2 with either HGS-ETR1 or HGS-ETR2, while DoHH2 was minimally inhibited by either antibody. Both HGS-ETR1 and HGS-ETR2 antibodies induced dose dependent increases in apoptosis (annexin V assay) of WSU-FSCCL and FC-TxFL-2, but not DoHH2. Caspase activation (flow cytometry using fluorescent substrates; Western analysis) was seen 4–6 hr after antibody addition. As expected, caspases 3 and 8 were activated by both antibodies in the two sensitive cell lines. Caspase 9, however, was also activated. Thus, TRAIL-R1 and TRAIL-R2 antibody binding inhibits growth, induces apoptosis and activates caspases 3, 8 and 9 in NHL cells expressing the targets. The induction of caspase 9 suggests cross-talk between the extrinsic and intrinsic pathways, in which activation of caspase 8 leads to cleavage and translocation of bid, which in turn leads to activation of caspase 9, and ultimately caspase 3. We have confirmed that bid cleavage does occur in these NHL cells after HGS-ETR1 and HGS-ETR2 binding. We have further shown that both the pan-caspase inhibitor ZVAD and a specific caspase 8 inhibitor block HGS-ETR1 and HGS-ETR2 induced apoptosis. As predicted, a specific inhibitor of caspase 9 only partially blocks apoptosis. This suggests that combination of the agonist HGS-ETR1 and HGS-ETR2 antibodies with agents that act via the caspase 9 pathway would be rational combinations to test for therapeutic potential in NHL.