Auxin-binding protein 1 (ABP1) is a receptor for the class of plant growth hormones designated auxins. ABP1 is a unique receptor because it resides primarily in the lumen of the endoplasmic reticulum (ER) however, two lines of evidence presented here suggest that ABP1 does not bind auxin within the endoplasmic reticulum, despite its predominant location there. First, ABP1 can not be photolabeled in intact cells that have accumulated the auxin and photolabeling reagent [7-³H], 5-azidoindole-3-acetic acid indicating either that auxin is excluded from the ER and is not available for photolabeling to ABP1 or that binding conditions within the ER lumen are insufficient for photolabeling. Second, at the pH of the ER lumen, auxin binding to ABP1 is not detectable. The pH estimate of the ER lumen is based on an indirect assay which indicates that the pH is closer to pH 7 than to the binding optimum of pH 5.5. These results support the hypothesis that ABP1 does not bind auxin within the ER and point to a site of action that is post ER. As a corollary, a second hypothesis on auxin binding to ABP1 in the ER lumen was tested and, by refuting this hypothesis, the results were found to be consistent with the above conclusion. This hypothesis, which is based on an observation by Napier and Venis (1990 Planta 182, 313-318) that auxin binding to purified ABP1 causes a protein conformational change at its putative ER-retention signal, is that auxin binding regulates the cellular location of ABP1 by inducing a masking of the ER-retention signal. Since this hormone-induced conformational change was shown with pure ABP1, implicit in our hypothesis is the concept that the structural requisites for this ligand-regulated translocation reside exclusively within the polypeptide. Therefore, to test this hypothesis, an animal expression system was chosen to exclude any plant specific contribution toward ABP1 trafficking. ABP1 expressed at high levels in mammalian cells (COS7) is efficiently retained in the ER lumen and is not secreted even in the presence of 190 uM indole-3-acetic acid, and auxin concentration that is 40 times above the K_d for IAA binding to ABP1.