We have developed an improved assay for detection of auxin-binding proteins using photoaffinity labeling. We have synthesized 5-NH2-[7-3H]indole-3-acetic acid (IAA) and 5-N3-[7-3H]IAA and extensively purified these compounds. Photoaffinity labeling of auxin binding proteins was performed at -196°C to reduce unspecific labeling of plant proteins. The method presented here has several advantages compared to previously described approaches. (1) Modification of the photoaffinity labeling conditions results in a significant increase in specificity of the assay; (2) the assay is simple and reliable; (3) the assay is fast and easily allows processing of a large number of samples. The application of this assay is demonstrated using a well known auxin-binding protein from maize coleoptiles, ABP-1. We show that ABP-1 is efficiently and specifically labeled after illumination in the presence of 5-N3-(7-3H]IAA. In addition, we demonstrate that photoaffinity labeling with 5-N3-[7-3H]IAA is specifically competed in the presence of an active auxin analogue. We conclude that the assay described should contribute to the identification of novel auxin-binding proteins.