Polarized sorting of rhodopsin in retinal rod photoreceptor cells is mediated by post-Golgi carrier membranes that bud from the trans-Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. The identity of the majority of the resident proteins of this organelle still remains elusive, despite multifaceted approaches to study this compartment. In the present study we have taken a proteomic approach to the analysis of the post-Golgi carriers. First, we modified the previously established fractionation protocols in order to achieve greater purity of the isolated membranes. Specifically, the new fractionation scheme depleted the post-Golgi fraction of cytosolic proteins that were the most abundant contaminants complicating analysis of two-dimensional (2-D) gel profiles in our previous preparations. The isolated membranes were subjected to 2-D gel electrophoresis, immunoblotting and microsequencing. This analysis showed that the improved subcellular fractionation yielded a fraction highly enriched in rhodopsin-bearing post-Gel carrier membranes. Two-dimensional mapping revealed 29 proteins that are preferentially found in this fraction and therefore represent candidates for post-Golgi membrane-specific proteins. This preparation of rhodopsin-bearing post-Golgi carriers is a first step towards the proteomics of this important organelle.