We have characterized the subcellular proteome of human embryonic stem cells (hESCs) through MS analysis of the membrane, cytosolic, and nuclear fractions, isolated from the same sample of undifferentiated hESCs. Strikingly, 74% of all proteins identified were detected in a single subcellular fraction; we also carried out immunofluorescence studies to validate the subcellular localization suggested by proteomic analysis, for a subset of proteins. Our approach resulted in deeper proteome coverage - peptides mapping to 893, 2475, and 1185 proteins were identified in the nuclear, cytosolic, and membrane fractions, respectively. Additionally, we used spectral counting to estimate the relative abundance of all cytosolic proteins. A large number of proteins relevant to hESC biology, including growth factor receptors, cell junction proteins, transcription factors, chromatin remodeling proteins, and histone modifying enzymes were identified. Our analysis shows that components of a large number of interacting signaling pathways are expressed in hESCs. Finally, we show that proteomic analysis of the endoplasmic reticulum (ER) and Golgi compartments is a powerful alternative approach to identify secreted proteins since these are synthesized in the ER and transit through the Golgi. Taken together, our results show that systematic subcellular proteomic analysis is a valuable tool for studying hESC biology. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.