Ubiquitin is covalently attached to target proteins by a multienzymatic system consisting of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligating) enzymes. NEDD8, a ubiquitin-like protein, is conjugated to proteins in a manner analogous to ubiquitinylation. beta-amyloid precursor protein-binding protein-1 (APPBP1) can bind to NEDD8 in rabbit reticulocyte lysates. However, since APPBP1 shows similarity to only the N-terminal domain of an E1 enzyme, it must interact with a protein showing similarity to the C-terminal region of E1s. By searching sequence databases, a cDNAs encoding UBA3 was identified as the human homolog of yeast Uba3. The predicted 442-amino acid UBA3 protein shares 43% sequence identity with yeast Uba3. In vitro, UBA3 formed a complex with APPBP1 and a thioester linkage with NEDD8. APPBP1/UBA3 complex may function as an E1-like enzyme for the activation of NEDD8.