Orexin-A and orexin-B are regulatory peptides involved in the control of feeding, sleep-wakefulness and exerting various endocrine and metabolic actions. Recently, we demontrated that orexins, acting at OX₁ receptor (OX₁R), are pro-apoptotic peptides. The aim of this study was to investigate the role of the receptor subtype OX₂R in the control of apoptosis. Orexins caused a caspase-dependant cell death by apoptosis and a drastic cell growth inhibition in Chinese hamster ovary (CHO) cells transfected with OX₂R cDNA. Upon addition of either orexin (10^-6M) for 48 h, apoptosis was demonstrated by DNA fragmentation, chromatin condensation, annexin-V binding and activation of caspase-3 and caspase-9. Orexins were active on apoptosis and cell growth inhibition in the range of concentrations between 10^-10 M and 10^-5M with an EC₅₀ of 5 x 10^-8M peptides. No effect of orexins could be detected in parental CHO cells. A rat pancreatic acinar cell line, AR42J, which expresses OX₂R but not OX₁R, also underwent growth suppression and apoptosis upon treatment with orexins. Suppression of AR42J cell growth by 10^-6M orexin was >75% after 24 h. Induction of annexin-V labeled AR42J cell number was dose-dependent with EC₅₀ of 5.1 x 10^-8M orexin-A and 9.8 x 10^-8M orexin-B. The OX₂R agonist [Ala¹¹, D-Leu¹⁵]orexin-B promoted effects on cell growth and apoptosis which were similar to those elicited by orexins. The OX₁R antagonist SB33487 did not alter orexin-induced inhibition of growth or orexin-induced stimulation of apoptosis in AR42J cells. For the first time, we provide functional and pharmacological evidence for a role of the OX₂R in orexin-induced apoptosis.