The mechanisms by which plants regulate levels of the phytohormone indole-3-acetic acid (IAA) are complex and not fully understood. One level of regulation appears to be the synthesis and hydrolysis of IAA conjugates, which function in both the permanent inactivation and temporary storage of auxin. Similar to free IAA, certain IAA-amino acid conjugates inhibit root elongation. We have tested the ability of 19 IAA-l-amino acid conjugates to inhibit Arabidopsis seedling root growth. We have also determined the ability of purified glutathione S-transferase (GST) fusions of four Arabidopsis IAA-amino acid hydrolases (ILR1, IAR3, ILL1, and ILL2) to release free IAA by cleaving these conjugates. Each hydrolase cleaves a subset of IAA-amino acid conjugates in vitro, and GST-ILR1, GST-IAR3, and GST-ILL2 have K(m) values that suggest physiological relevance. In vivo inhibition of root elongation correlates with in vitro hydrolysis rates for each conjugate, suggesting that the identified hydrolases generate the bioactivity of the conjugates.