Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p < 0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y, Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.