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To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B 2 bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cytdel-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B 2 antagonist HOE 140. B 2 was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B 2 and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B 2 receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B 2 receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.Renin was discovered over a century ago (1), and kallikrein was discovered about 25 years later (2). Many of the cascading events initiated by these proteases are integrated by angiotensin I-converting enzyme (ACE) 1 or kininase II (3) as it activates angiotensin I to angiotensin II (4) and inactivates kinins (5).Subsequently, it became obvious that inhibitors of ACE affect the metabolism of both peptides (6). The successful clinical applications of ACE inhibitors have gone far beyond controlling elevated blood pressure (7, 8), but questions remain regarding which of the beneficial effects are due to inhibiting angiotensin II activation and which are caused by blocking the enzymatic breakdown of bradykinin (BK) or kallidin. The very extensive clinical applications of ACE inhibitors, not only in treating hypertension but also in treating cardiac conditions, (e.g. congestive heart failure or after myocardial infarction), and in diabetic nephropathies (9 -11), have kept attention focused on this issue. In laboratory experiments and in some clinical studies (12, 13), many effects of ACE inhibitors were abolished by the BK B 2 receptor blocker HOE 140. Although it was assumed that these effects were due to inhibiting the inactivation of BK, early bioassays already indicated that substances that did not prolong the half-life of BK still potentiated its actions on the isolated guinea pig ileum (14). Experiments on isolated guinea pig atria demonstrated that ACE inhibitors can resensitize the heart tissue desensitized by a B 2 receptor agonist (15). ...