Publisher Summary Opsins and 11-cis-retinaldehyde (RAL) form the visual pigments in the photoreceptor cells of the eye. The photoreceptor cells are mainly supplied with 11-cis-RAL by the underlying retinal pigment epithelium (RPE). The enzymatic activity responsible for the oxidation of 11-cis-ROH is microsomal. To delineate the complex retinoid-metabolizing pathways in the RPE, and the interplay between the RPE and the adjacent photoreceptor cells, it is important to investigate the cell biology of retinoid processing. This chapter describes several classic techniques to study the topology of membrane proteins and to determine the membrane topology of 11-cis-RDH. Experiments were conducted by applying a protease protection technique to both RPE-derived microsomes and heterologous microsomes containing 11-cis-RDH generated by in vitro translation. As an independent method to determine the membrane topology of 11-cis-RDH, the ability of mutants of 11-cis-RDH, containing consensus acceptor sites for N-linked glycosylation, is also examined.