The Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) interact with a group of serinethreonine protein kinases (AtCIPKs) in a calciumdependent manner. Here we identify a 24 amino acid domain (NAF domain) unique to these kinases as being required and suf®cient for interaction with all known AtCBLs. Mutation of conserved residues either abolished or signi®cantly diminished the af®nity of AtCIPK1 for AtCBL2. Comprehensive two-hybrid screens with various AtCBLs identi®ed 15 CIPKs as potential targets of CBL proteins. Database analyses revealed additional kinases from Arabidopsis and other plant species harbouring the NAF interaction module. Several of these kinases have been implicated in various signalling pathways mediating responses to stress, hormones and environmental cues. Full-length CIPKs show preferential interaction with distinct CBLs in yeast and in vitro assays. Our ®ndings suggest differential interaction af®nity as one of the mechanisms generating the temporal and spatial speci®city of calcium signals within plant cells and that different combinations of CBL±CIPK proteins contribute to the complex network that connects various extracellular signals to de®ned cellular responses.