In birds, adrenocorticotropin (ACTH) release from the anterior pituitary gland during stress is controlled by corticotrophin-releasing hormone (CRH) and arginine vasotocin (AVT). Using five-week old male chicks, simultaneous intravenous injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared to that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRH and VT2 receptors formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18% and the distance between receptors was 5-6 nm. Treatment of the cells which expressed both CFP-CRHR and YFP-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRH and VT2 receptors, treatment with CRH and AVT resulted in a significant increase in cyclic AMP production over that with CRH alone indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.