In eukaryotes, members of the Ero1 family control oxidative protein folding in the endoplasmic reticulum (ER). Yeast Ero1p is tightly associated with the ER membrane, despite cleavage of the leader peptide, the only hydrophobic sequence that could mediate lipid insertion. In contrast, human Ero1-Lalpha and a yeast mutant (Ero1pDeltaC) lacking the 127 C-terminal amino acids are soluble when expressed in yeast. Neither Ero1-Lalpha nor Ero1pDeltaC complements an ERO1 disrupted strain. Appending the yeast C-terminal tail to human Ero1-Lalpha restores membrane association and allows growth of ERO1 disrupted cells. Therefore, the tail of Ero1p mediates membrane association and is crucial for function.