The large-conductance voltage- and Ca2+-activated K+ channels (BK) are activated by membrane depolarizations and intracellular Ca2+. Two consecutive intracellular RCK (Regulators of K+ Conductance) domains, RCK1 and RCK2, are responsible for calcium sensitivity and together form the majority of the C-terminus.Information about the structural and functional properties of the individual RCK1 and RCK2 domains has been reported by our group (Yusifov, et. al. PNAS 2008). However, the characterization of the full C-terminus remains unresolved. Here we report the initial structural and functional characterization of a high-purity protein expressed and purified from E. coli corresponding to the human (hSlo) BK channel C-terminus. The expressed C-terminus includes 684aa, starting from the S6-RCK1 linker and encompassing RCK1, RCK2, and the interconnecting 92aa RCK1-RCK2 linker.The calcium-binding activity of the C-terminus (10ug), loaded on a nitrocellulose membrane, was probed by dot blot analysis of 45Ca2+-binding. The C-terminus displayed a strong calcium-binding property when compared to Albumin.The organization of the secondary structure of the C-terminus was investigated using Circular Dichroism (CD) spectroscopy. Far-UV CD spectra (190-260nm) of the C-terminus, analyzed with CONTIN/LL algorithm from the CDpro suite (SMP56 protein reference set), gave a secondary structure consisting of 29% α-helix, 20% β-strand, 22% turn, and 29% unordered.The quaternary structure of C-terminus was investigated using size-exclusion chromatography with a Superdex 200 10/300 column. The C-terminus eluted in a single peak at a molecular weight of 330kDa corresponding to the theoretical tetrameric C-terminus complex (310kDa). In denaturating condition (SDS-gel electrophoresis), the C-terminus migrated as a monomeric 74kDa band (expected 77.6kDa).In conclusion, we have successfully purified the functional human BK channel C-terminus domain, which allows for further investigation of the properties of the mammalian BK “Gating ring,” encompassing eight RCK domains.