Ubiquitin-activating enzyme, E1, directs the ATP-dependent formation of a thiol ester linkage between itself and ubiquitin. The energy in this bond is ultimately used to attach ubiquitin to various intracellular proteins. We previously reported the isolation of multiple E1s from wheat and the characterization of a cDNA encoding this protein (UBA1). We now report the derived amino acid sequence of two additional members of this gene family (UBA2 and UBA3). Whereas the amino acid sequence of UBA2 is nearly identical to UBA1, the sequence of UBA3 is significantly different. Nevertheless, the protein encoded by UBA3 catalyzes the ATP-dependent activation of ubiquitin in vitro. Comparison of derived amino acid sequences of genes encoding E1 from plant, yeast, and animal tissues revealed 5 conserved cysteine residues, with one potentially involved in thiol ester bond formation. To identify this essential residue, codons corresponding to each of the 5 cysteines in UBA1 were individually altered using site-directed mutagenesis. The mutagenized enzymes were expressed in Escherichia coli and assayed for their ability to activate ubiquitin. Only substitution of the cysteine at position 626 abolishes E1 activity, suggesting that this residue forms the thiol ester linkage with ubiquitin.