The tight coupling between ongoing translation and translocation across the mammalian endoplasmic reticulum has made it difficult to determine the requirements that are specific for translocation. We have developed an in vitro assay that faithfully mimics the co-translational targeting and translocation of the amino terminus of opsin without ongoing translation. Using this system we demonstrate that this post-translational targeting and translocation requires nucleotide triphosphates but not cytosolic proteins. The addition of GTP alone was sufficient to fully restore targeting. The addition of ATP was not specifically required, and non-hydrolyzable analogs of ATP that blocked 90% of the ATPase activity also had no inhibitory effect on translocation.