An analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg−1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg−1 FW compared to approximately 15 pg mg−1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.