We designed and synthesized new photoactivatable linear vasopressin analogues containing benzophenone photophores. All compounds were monitored and purified using RP-HPLC and characterized by mass spectrometry. Affinity and selectivity were determined in CHO cells expressing either human V(1a), V(1b) or V(2) receptor subtypes. Within the series, compounds 6 (PhCH(2)CO-lBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH(2)) and 9 (PhCH(2)CO-dBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH(2)), containing a benzoylphenylalanine residue (Bpa), were selected and their antagonistic properties determined (K(inact) = 1.87 and 0.35 nM, respectively). The dissociation constant of the most potent candidate (compound 9) was further calculated from saturation experiments using the (125)I derivative (K(d) = 0.07 +/- 0.01 nM). Photolabeling experiments using radioactive compound 9 as a probe were specific and UV-dependent and allowed the identification of two bands at molecular masses around 85-90 kDa and 46 kDa, respectively, as previously described by Phalipou et al., using two photoreactive linear azidopeptide antagonists. The results suggest therefore that compound 9 is a potent new tool for the accurate mapping of the human V(1a) receptor antagonist binding site.