Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics.