A novel modular design is presented for the introduction of biotinylated photoprobes containing either 4-azidotetrafluorobenzamide, 4-(1-azi-2,2,2-trifluoroethyl)benzamide, or 4-benzoylbenzoylamide. The use of biotinylated affinity labels offers several advantages over radiolabeled probes by virtue of their exploitation of the biotin-avidin system of detection and purification. A biotinylated benzoylbenzoyl photoprobe of pepstatin (BBB-pepstatin, 5) was synthesized in three steps from pepstatin. The photoprobe is a competitive inhibitor of porcine pepsin, with an apparent dissociation constant of 31 pM. Western blotting of BBB-pepstatin-photolabeled porcine pepsin, renin, cathepsin D and human renin, and cathepsin D could be detected with an avidin-horseradish peroxidase label. Routinely, 7 pM of aspartic protease could be photolabeled and detected with this system. The pepstatin photoaffinity probe is also very selective; the probe failed to label cysteine protease (papain), metalloprotease (carboxypepitase A), and serine protease (chymotrypsin and trypsin). To further establish the utility of the biotinylated probe, BBB-pepstain-photolabeled porcine pepsin was purified by monomeric avidin chromatography. This probe should be useful for the identification of unknown cytosolic and membrane-bound aspartic proteases.