The photoaffinity labeling agent azido-IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analogue of the endogenous auxin indole-3-acetic acid, was used to search for auxin-binding proteins in the soluble fraction of Hyoscyamus muticus cells. Azido-IAA became covalently attached to three polypeptides with a high specific activity. The labeling was specific for IAA and not due to random tagging. Two polypeptides with molecular masses of 31 and 24 kDa in the 0-30% ammonium sulfate fraction were labeled after UV photolysis at 0 degree C but not at -196 degrees C, and appeared to have a high affinity indole-binding site(s) for which active, non-indole auxins were not good ligands. A third polypeptide with a molecular mass of 25 kDa present in the 50-60% ammonium sulfate fraction labeled exclusively at -196 degrees C and had a significant affinity for active auxins but not for inactive indoles. The azido-IAA labeling pattern, pI, competition results, and immunoprecipitation all indicate that the 31- and 24-kDa polypeptides are related to the basic form of endo-1,3-beta-glucanase (EC 3.2.1.39). Azido-IAA labeling polypeptides equivalent to the 31- and 24-kDa species were apparently also present in the cell wall. The low pH optimum for binding of azido-IAA to the 25-kDa polypeptide suggests the location of the active protein in a compartment such as the vacuole or a transport vesicle rather than in the cytosol.