Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.