A neutral proteolytic activity that converts human Big endothelin-1 (Big Et-1) to endothelin-1 has been identified from a human endothelial hybrid cell line, EAHY 926. This enzyme is an integral membrane protein and cofractionates with other enzymes typically found in the plasma membrane. The activity has been solubilized with nonionic detergents and purified 1000-fold by a combination of lectin affinity chromatography, ion-exchange chromatography, and chromatography on red-dye agarose. The partially purified activity is a metalloenzyme based upon its sensitivity to chelating agents, competitive inhibition by phosphoramidon, and reconstitution with ZnCl2 or CoCl2 following EDTA inactivation. The enzyme appears to be unique, however, as it is not inhibited by specific inhibitors of known metalloproteases. It correctly processes Big Et-1 to Et-1 and the complementary C-terminal fragment with a sharp pH optimum near 7.0. Both the Km for Big Et-1 and the Ki for phosphoramidon are pH-dependent, with values of 5-7 and 3.5 microM, respectively, at pH 7.0. The enzyme also cleaves Big Et-2 with a Km of 27.9 microM and a Vmax one-third that for Big Et-1 but has no appreciable activity toward Big Et-3. An s20,w of 9.5 S was determined by sucrose density ultracentrifugation in H2O and D2O. When combined with a Stokes radius of 56 A determined by gel filtration, the enzyme had a calculated apparent molecular weight of 250,000. Conditions have been established to renature the activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions activity was detected in a protein band at 280 kDa, in agreement with the aforementioned molecular weight determination. From these results, a kcat/Km of 1 x 10(6) M-1 s-1 was estimated for the purified enzyme with Big Et-1 as a substrate, which is a reasonable value for a protease acting upon its physiologic substrate. Several criteria indicate that the activity isolated from EAHY cells is the physiologically relevant endothelial-derived endothelin converting enzyme. On the basis of our results, this enzyme is present in low abundance in endothelial cells and at least a 100,000-fold purification will be required to obtain a homogeneous preparation. However, because EAHY cells can be grown in large numbers, they can supply the quantities of enzyme required both for biochemical studies and for the development of specific inhibitors.