Angiotensin-Converting Enzyme 2 (ACE2) is a homologue of the well-characterised plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus. We have recently shown that like Angiotensin Converting Enzyme (ACE), ACE2 undergoes ectodomain shedding and that this shedding event is upregulated by phorbol esters. In the present study, we have used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16 amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to GST-calmodulin, but not GST alone, in pull-down assays using cell lysates. Finally, to investigate if calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin specific inhibitors; trifluoperazine (TFP) and calmidazolium. Both TFP (25 micromol/L) and calmidazolium, (25 micromol/L), significantly increased the release of ACE2 into the medium (44.1+/-10.8 p <0.05, Student s t-test; unpaired, two-tailed, and 51.1+/-7.4 p <0.05, One-way ANOVA, respectively;), as analysed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin specific inhibitor -stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.