Escherichia coli (strain K-12)-soluble proteins were analyzed by nondenaturing micro 2-DE and MALDI-MS-PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939-948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB-stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2-DE gels (one for wide mass range and one for low-molecular-mass range) were numbered and subjected to MALDI-MS-PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p < 0.05) with polypeptides in Swiss-Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2-DE gels and the locations of the activity spots matched well with those of the MS-assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss-Prot (119 polypeptides as homo-multimers and 25 as hetero-multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2-DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third-dimension micro SDS-PAGE (micro 3-DE), about 190 CBB-stained spots were detected. The assignment of the polypeptides separated on the 3-DE gel would further provide information on protein/polypeptide interactions.