The Ca²⁺-stimulated adenylyl cyclase type 8 (AC8) generates dynamic patterns of cAMP signal in response to local Ca²⁺ events to coordinate the actions of Ca²⁺ and cAMP (1). Previous studies have revealed that AC8 is uniquely sensitive to sub-µM Ca²⁺ rises mediated by capacitative Ca²⁺ entry (CCE). In contrast, AC8 displays little sensitivity to Ca²⁺ release from ER stores and to other forms of Ca²⁺ entry, including ionophore-mediated entry (2). Part of the selectivity of AC8 for CCE is thought to rely on its targeting to lipid rafts alongside CCE channels (3). Here, we have tethered a genetically-encoded Ca²⁺ sensor, GCaMP2 (4), to the N-terminus of AC8 to directly monitor Ca²⁺ changes within the immediate vicinity of the AC when exposed to a range of Ca²⁺ stimuli in HEK293 cells. Comparisons were made between the GCaMP2-AC8 sensor and GCaMP2-AC2 (AC2 is a Ca²⁺-insensitive, non-raft targeted AC). All experiments were performed at RT using a CCD-camera imaging system. In situ calibrations provided Kd values of 316nM for GCaMP2-AC8 and 240nM for GCaMP2-AC2 with Hill coefficients ~3.0 (consistent with previous data for untagged GCaMP2 (4)). To compare the response of the targeted sensors to different modes of Ca²⁺ increase the muscarinic agonist, carbachol, was used to trigger ER store depletion and CCE was then evoked upon addition of 2mM external Ca²⁺. GCaMP2-AC2 detected a 7.26 ± 0.91 fold larger Ca²⁺ signal during IP₃-mediated Ca²⁺ release than during CCE (n=34 cells). In GCaMP2-AC8 expressing cells this ratio was decreased to 1.73 ± 0.20 (n=87; p<0.01) with ~50% of cells detecting no Ca²⁺ rise during IP₃-mediated release. Furthermore, the rate of signal increase during CCE was faster for GCaMP2-AC8 (26.8 ± 2.0s to peak) than for GCaMP2-AC2 (77.4 ± 5.2s to peak) suggesting that AC8 resides much closer to sites of CCE. Consistent with this hypothesis pre-incubation with 10µM EGTA-AM did not significantly reduce the initial CCE-induced Ca²⁺ rise detected by GCaMP2-AC8 (n=40). In contrast, detection of CCE by GCaMP2-AC2 was attenuated by EGTA (n=15; p<0.01). Neither sensor detected any Ca²⁺ rise following BAPTA loading (n=16). Finally, we examined whether GCaMP2-AC8 could detect ionophore-mediated Ca²⁺ entry. In all HEK293 cells tested, GCaMP2-AC8 was unable to detect the non-specific Ca²⁺ entry during ionomycin treatment (n=75). Our findings suggest that AC8 resides in a discrete microdomain that is exposed to rapid Ca²⁺ changes during CCE but is uniquely ‘shielded’ from other modes of Ca²⁺ rise. Differential distribution of AC8 and AC2 into sub-domains of the plasma membrane that differ, not only in terms of their lipid composition (raft vs. non-raft), but also by their exposure to distinct local Ca²⁺ signals, serves to optimise interplay between dynamic Ca²⁺ events and AC8-mediated cAMP production.