AbstractIntracellular Ca2+signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF‐hand Ca2+‐binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS‐1 and neurocalcin δ were prepared as N‐terminally tagged GST fusion proteins, and the less closely related protein L‐CaBP1 was prepared in both N‐ and C‐terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 μM free Ca2+. Bound proteins were eluted with Ca2+‐free and high‐salt buffers and eluted proteins were identified by MALDI‐MS and Western blotting. New protein targets detected included ARF1, Ca2+‐dependent activator protein for secretion 1, cyclic nucleotide 3', 5'‐phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF‐β receptor. While certain of these interactions occurred with more than one of the Ca2+‐binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin‐binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.