Activation of OX₁ orexin receptors heterologously expressed in CHO cells led to a rapid, strong and long-lasting increase in ERK phosphorylation (activation). Dissection of the signal pathways to ERK using multiple inhibitors and dominant-negative constructs indicated involvement of Ras, protein kinase C, phosphoinositide-3-kinase and Src. Most interestingly, Ca²⁺ influx appeared central for the ERK response in CHO cells, and the same was indicated in recombinant neuro-2a cells and cultured rat striatal neurons. Detailed investigations in CHO cells showed that inhibition of the receptor- and store-operated Ca²⁺ influx pathways could fully attenuate the response, whereas inhibition of the store-operated Ca²⁺ influx pathway alone or the Ca²⁺ release were ineffective. If the receptor-operated pathway was blocked, exogenously activated store-operated pathway could take its place and restore the coupling of OX₁ receptors to ERK. Further experiments suggested that Ca²⁺ influx as such may not be required for ERK phosphorylation, but that Ca²⁺, elevated via influx, acts as a switch enabling OX₁ receptors to couple to cascades leading to ERK phosphorylation, cAMP elevation and phospholipase C activation. In conclusion, the data suggest that the primary coupling of orexin receptors to Ca²⁺ influx allows them to couple to other signal pathways; in the absence of coupling to Ca²⁺ influx, orexin receptors can act as signal integrators by taking advantage of other Ca²⁺ influx pathways.