Identification of membrane proteomes remains challenging. Here, we present a simple, fast, and scalable off-line procedure based on three-phase partitioning with butanol to fractionate membrane proteomes in combination with both in-gel and in-solution digestions and mass spectrometry. This should help to further accelerate the field of membrane proteomics. Using this new strategy, we analyzed the salt-stripped thylakoid membrane of chloroplasts of Arabidopsis thaliana. 242 proteins were identified, at least 40% of which are integral membrane proteins. The functions of 86 proteins are unknown; these include proteins with TPR, PPR, rhodanese, and DnaJ domains. These proteins were combined with all known thylakoid proteins and chloroplast (associated) envelope proteins, collected from primary literature, resulting in 714 non-redundant proteins. They were assigned to functional categories using a classification developed for MapMan (Thimm, O., Blasing, O., Gibon, Y., Nagel, A., Meyer, S., Kruger, P., Selbig, J., Muller, L. A., Rhee, S. Y., and Stitt, M. (2004) Plant J. 37, 914-939), updated with information from primary literature. The analysis elucidated the likely location of many membrane proteins, including 190 proteins of unknown function, holding the key to better understanding the two membrane systems. The three-phase partitioning procedure added a new level of dynamic resolution to the known thylakoid proteome. An automated strategy was developed to track possible ambiguous identifications to more than one gene model or family member. Mass spectrometry search results, ambiguities, and functional classifications can be searched via the Plastid Proteome Database.