Mutations in the AXR1 gene result in a reduction in auxin response and diverse defects in auxin-regulated growth and development. In a previous study, we showed that AXR1 forms a heteradimer with the ECR1 protein. This enzyme activates the ubiquitin-related protein RUB1 in vitro. Furthermore, we showed that the Skp1-Cul1/Cdc53-F-bax (SCF) subunit AtCUL1 is modified by RUB1 in viva. In this report, we demonstrate that the formation of RUB-AtCUL1 is dependent on AXR1 and ECR1 in viva. The expression of AXR1 and ECR1 is restricted to zones of active cell division and cell elongation, consistent with their role in growth regulation. These results provide strong support for a model in which RUB conjugation of AtCUL1 affects the function of SCF E3s that are required for auxin response.