Ca2+ is a ubiquitous signaling messenger mediating many essential cellular functions such as excitability, exocytosis and transcription. Among the different pathways by which cellular Ca2+ signals are generated, the entry of Ca2+ through store-operated Ca2+ release-activated Ca2+ (CRAC) channels has emerged as a widespread mechanism for regulating Ca2+ signaling in many eukaryotic cells. CRAC channels are implicated in the physiology and pathophysiology of numerous cell types, underlie several disease processes including a severe combined immunodeficiency syndrome, and have emerged as major targets for drug development. Although little was known of the molecular mechanisms of CRAC channels for several decades, the discovery of Orai1 as a prototypic CRAC channel pore-subunit, and the identification of STIM1 as the ER Ca2+ sensor, have led to rapid progress in our understanding of many aspects of CRAC channel behavior. This review examines the molecular features of the STIM and Orai proteins that regulate the activation and conduction mechanisms of CRAC channels.