Auxins are phytohormones that are essential for many aspects of plant growth and development. The main auxin produced by plants is indole-3-acetic acid (IAA). IAA exists in free and conjugated forms, corresponding to the bioactive and stored hormones, respectively. Free IAA levels, which are crucial for various physiological activities, are maintained through a complex network of environmentally and developmentally responsive pathways including IAA biosynthesis, transport, degradation, conjugation, and conjugate hydrolysis. Among conjugated IAA forms, ester- and amide-type conjugates are the most common. Here we identify a new gene, UPRIGHT ROSETTE (URO), the overexpression of which alters IAA homeostasis in Arabidopsis (Arabidopsis thaliana). We previously identified a semidominant mutant, uro, which had multiple auxin-related phenotypes. We show here that compared to wild-type plants, the uro plants contain increased levels of free and ester-conjugated IAA, and decreased levels of amino-conjugated IAA. uro plants carrying the pDR5:beta-glucuronidase (GUS) construct have strong GUS staining in cotyledons and stem, and their cotyledons are able to generate roots on auxin-free medium, further confirming that this mutant contains higher levels of free IAA. The URO gene encodes a C2H2 zinc-finger protein that belongs to a plant-specific gene family. The response to URO overexpression is evolutionarily conserved among plants, as GUS activity that may reflect free IAA levels was increased markedly in transgenic p35S:URO/pGH3:GUS/Physcomitrella patens and pNOS:URO/pGH3:GUS/P. patens plants.