The known structure-activity relationship and docking models for peptide ligands of the bradykinin B(2) receptor indicate a certain tolerance to N-terminal extension. We took advantage of this by generating two fluorescent bradykinin analogs containing 5(6)-carboxyfluorescein (CF) optionally used with the ε-aminocaproyl spacer condensed at the N terminus of the agonist. Pharmacological studies indicated that CF-bradykinin was virtually inactive as a B(2) receptor ligand and agonist, whereas CF-ε-aminocaproyl-bradykinin (CF-εACA-BK) was 400- to 1000-fold less potent than bradykinin (competition of [(3)H]bradykinin binding to B(2) receptors, contractility of the human isolated umbilical vein). Nevertheless, CF-εACA-BK (5 μM) was taken up by human embryonic kidney 293a cells expressing recombinant B(2) receptors, but not by those cotreated with an antagonist or expressing a truncated receptor that is pharmacologically intact but not phosphorylable. A higher-affinity CF-conjugated peptide, the antagonist CF-εACA-d-Arg-[Hyp(3),Igl(5),d-Igl(7),Oic(8)]-bradykinin (B-10380), labeled both intact and truncated receptor forms at the cell surface. The fluorescent agonist CF-εACA-BK was found in vesicles positive for β-arrestin(1), Rab5, and Rab7, then apparently degraded as a function of time because the fluorescence was transferred from the vesicles to the cytosol in a vesicular-ATPase-dependent process (3 h). The ectopeptidase angiotensin-converting enzyme (ACE) is a major kininase. The binding affinity of CF-εACA-BK for this carboxydipeptidase is identical to that of bradykinin ([(3)H]enalaprilat displacement assay). Recombinant ACE is essentially a plasma membrane protein in CF-εACA-BK imaging of intact cells. Micromolar CF-εACA-BK is a probe for the two major physiological targets of bradykinin, the B(2) receptor and ACE. As an agonist, it is subjected to β-arrestin-mediated endocytosis, trafficking, and subsequent ligand degradation.