GRAB(ACh3.0) is a genetically encoded fluorescent sensor for acetylcholine that can be used for real-time detection of endogenous acetylcholine dynamics. It is a G-protein-coupled receptor activation-based ('GRAB') sensor based on the acetylcholine receptor encoded by the Homo sapiens CHRM3 gene (HGNC:1952) and containing a circularly permuted form of EGFP (cpEGFP) inserted into an intracellular loop. GRAB(ACh3.0) was derived from GRAB(ACh2.0) using site-directed mutagenesis and shows significantly improved sensitivity and minimal coupling to major GPCR-mediated downstream signaling pathways (FBrf0247870).