Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated1,4- benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H- benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo- 5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK 1 and CCK 2) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The 125I-labeled CCK 1 receptor-selective compound 9 often revealed a substantially higher amount of CCK 1 receptor binding sites in tumors than the agonist 125I-CCK. Conversely, the radioiodinated CCK 2 receptor-selective compound 7 showed generally weaker tumor binding than 125I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK 1 receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK 1 receptor-expressing tumors in vivo.