Microsomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msA.LDH fusion protein containing the last 35 amino acids of msA-LDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37degreesC. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4degreesC, the recombinant GFPALDH(35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPA.LDH(35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.