Ubiquitination affects diverse physiological processes in eukaryotic cells. AtRMA1 was previously identified as an Arabidopsis homolog of human RING membrane-anchor E3 ubiquitin (Ub) ligase. Here, we identified two additional AtRMA homologs, AtRMA2 and AtRMA3. The predicted AtRMA proteins contain a RING motif and a trans-membrane domain in their N-terminal and extreme C-terminal regions, respectively. Bacterially expressed AtRMAs exhibited E3 ligase activity in vitro, which was abrogated by mutation of the conserved cysteine residue in their RING domains. In vivo targeting experiments using an Arabidopsis protoplast-transfection system showed that all three AtRMAs are localized to the ER. Although RT-PCR analysis indicated that AtRMA mRNAs were expressed constitutively in all tissues examined, their promoter activities were differentially detected in a tissue-specific fashion in AtRMA-promoter::GUS transgenic Arabidopsis plants. The AtRMA1 and AtRMA3 genes are predominantly expressed in major tissues, such as cotyledons, leaves, shoot-root junction, roots, and anthers, while AtRMA2 expression is restricted to the root tips and leaf hydathodes. We suggest that a ubiquitnation pathway involving these AtRMA E3 Ub ligases may play a role in the growth and development of Arabidopsis.