A variety of assays have been developed which permit rapid and unambiguous determination of the membrane topology adopted by newly synthesized proteins. Cell-free systems and microinjected Xenopus oocytes are two of the most attractive approaches for characterizing the elements in both the nascent polypeptide and the membrane which together determine the final orientation of the protein in the membrane. Careful analysis of the mechanism of protein translocation using these methods has revealed a number of unusual topologies. The applications of a number of different assays for endoplasmic reticulum membrane translocation are described for the most commonly used cell-free systems (wheat germ and reticulocyte lysate), as well as for microinjected Xenopus oocytes.