Angiotensin-converting enzyme 2 (ACE2) is a recently described member of the 
renin-angiotensin system that hydrolyzes angiotensin (Ang) II to Ang-(1-7), and 
may thereby protect against cardiovascular and renal diseases. ACE2 is a type 1 
integral membrane protein and contains a catalytically active ectodomain that 
can be shed from the cell surface into the extracellular space, via cleavage by 
a disintegrin and metalloproteinase-17 (ADAM-17). ACE2 enzymatic activity and 
protein can be detected in biological fluids, including urine, plasma, and 
conditioned cell culture media. We present a detailed method for measurement of 
ACE2 activity in biological fluids, using hydrolysis of an intramolecularly 
quenched fluorogenic ACE2 substrate, in the absence or presence of the ACE2 
inhibitors MLN-4760 or DX600. Recombinant human or mouse ACE2 is used to 
generate standard curves for this assay, with ACE2 detection ranging from 1.56 
to 50 ng/ml. While MLN-4760 potently inhibits the activity of both human and 
mouse ACE2, DX600 (linear form) only effectively blocks human ACE2 activity in 
this assay. In biological samples of human and mouse urine, cell culture medium 
from mouse proximal tubular cells, and mouse plasma, the mean intra- and 
inter-assay coefficients of variation (CVs) of the assay range from 1.43 to 4.39 
%, and from 7.01 to 13.17 %, respectively. We present data on the time and 
substrate concentration dependence of the assay, and show that exogenous D 
-glucose, creatinine, urea, and albumin do not interfere with its performance. 
In biological fluids, this assay is a simple and reliable method to study the 
role of ACE2 and its shed fragments in cardiovascular and renal diseases.