Unlike the widely distributed and preformed B(2) receptors, the bradykinin B(1) receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B(1) receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-epsilon-aminocaproyl-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-bradykinin] and the agonist B-10378 [5(6)CF-epsilon-aminocaproyl-Lys-des-Arg(9)-bradykinin]. B-10376 has a K(i) of 10 to 20 nM to displace [(3)H]Lys-des-Arg(9)-bradykinin from rabbit or human recombinant B(1) receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA(2), 7.49). B-10378 was a full agonist at the naturally expressed B(1) receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition K(i) of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B(1) receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B(1) receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B(1) receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B(1) receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).