Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca2+ indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480 nm/535 nm was repeatedly increased when the intracellular Ca2+ concentration ([Ca2+](i)) was raised. F2C had a Ca2+ sensitivity with an apparent dissociation constant (K-d for Ca2+) of 150 nM, and demonstrated kinetics that paralleled Fura-2 when [Ca2+](i) was measured simultaneously. These properties of F2C are useful to be a Ca2+ indicator. (c) 2005 Elsevier Inc. All rights reserved.