Background and PurposeOne of the major responses upon orexin receptor activation is Ca2+ influx, and this influx seems to amplify the other responses mediated by orexin receptors. However, the reduction in Ca2+, often used to assess the importance of Ca2+ influx, might affect other properties, like ligand−receptor interactions, as suggested for some GPCR systems. Hence, we investigated the role of the ligand−receptor interaction and Ca2+ signal cascades in the apparent Ca2+ requirement of orexin‐A signalling.Experimental ApproachReceptor binding was assessed in CHO cells expressing human OX1 receptors with [125I]‐orexin‐A by conventional ligand binding as well as scintillation proximity assays. PLC activity was determined by chromatography.Key ResultsBoth orexin receptor binding and PLC activation were strongly dependent on the extracellular Ca2+ concentration. The relationship between Ca2+ concentration and receptor binding was the same as that for PLC activation. However, when Ca2+ entry was reduced by depolarizing the cells or by inhibiting the receptor‐operated Ca2+ channels, orexin‐A‐stimulated PLC activity was much more strongly inhibited than orexin‐A binding.Conclusions and ImplicationsCa2+ plays a dual role in orexin signalling by being a prerequisite for both ligand−receptor interaction and amplifying orexin signals via Ca2+ influx. Some previous results obtained utilizing Ca2+ chelators have to be re‐evaluated based on the results of the current study. From a drug discovery perspective, further experiments need to identify the target for Ca2+ in orexin‐A−OX1 receptor interaction and its mechanism of action.