In a previous paper, we reported on the analysis of Escherichia coli (strain K-12) soluble proteins by nondenaturing micro 2-DE/3-DE and MALDI-MS-PMF [Manabe, T., Jin, Y., Electrophoresis 2010, 31, 2740-2748]. To evaluate the performance of the 2-DE/3-DE technique, a nondenaturing 2-DE gel just after the second-dimension run was cut into 12 vertical strips, each 2 mm-wide strip was set on a micro slab gel, and third-dimension SDS-PAGE was run in parallel. Each of the twelve 3-DE gels showed about 150-200 CBB-stained spots. Two of the 3-DE gels were selected for the assignment of polypeptides using MALDI-MS-PMF and totally 161 polypeptides were assigned on the two 3-DE gels, in which 81 have been assigned on the nondenaturing micro 2-DE gel and 80 were newly assigned. Most of the newly assigned polypeptides resided in faintly stained spots on the 3-DE gels, which indicates that the polypeptides were purified in the process of the third-dimension separation. The comparisons of the apparent mass values estimated from the second-dimension (nondenaturing pore-gradient PAGE) mobility with those estimated from the third-dimension (SDS-PAGE) mobility suggested the oligomer structures of the assigned polypeptides and they matched well with those described in a database (UniProtKnowledgebase). The technique of nondenaturing micro 2-DE/3-DE, combined with MALDI-MS-PMF, could become an efficient method to obtain information on the quaternary structures of hundreds of cellular soluble proteins simultaneously because of its high efficiency in protein/polypeptide separation and assignment.