Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B₂ receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B₂ receptors directly. To exclude intermediate kinin release or kininogen uptake from the culture medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected CHO cells that express human B₂ receptors (CHO-B₂) and cells that co-express angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated or baseline levels. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. Next, we tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analogue as a control for these experiments. Enalaprilat (1 µM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B₂ cells that do not bear ACE. We concluded that kallikrein activated B₂ receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.