We have combined sucrose density gradient subcellular fractionation with quantitative, tandem-mass-spectrometry-based shotgun proteomics to investigate spatial distributions of proteins in MCF-7 breast cancer cells. Emphasis was placed on four major organellar compartments: cytosol, plasma membrane, endoplasmic reticulum, and mitochondrion. Two-thousand one-hundred eighty-four proteins were securely identified. Four-hundred eighty-one proteins (22.0% of total proteins identified) were found in unique sucrose gradient fractions, suggesting they may have unique subcellular locations. 454 proteins (20.8%) were found to be ubiquitously distributed. The remaining 1249 proteins (57.2%) were consistent with intermediate distribution over multiple, but not all, subcellular locations. Ninety-four proteins implicated in breast cancer and 478 other proteins which share the same five major cellular biological processes with a majority of the breast cancer proteins were observed in 334 and 1223 subcellular locations, respectively. The data obtained is used to evaluate the possibility of defining more exact sets of subcellular organelles, the completeness of current descriptions of spatial distribution of cellular proteins, the importance of multiple subcellular locations for proteins in functional processes, the subcellular distribution of proteins related to breast cancer, and the possibility of using these methods for dynamic spatio/temporal studies of function/regulation in MCF-7 breast cancer cells.