Although a great deal is known about the structure and function of most mammalian organelles, comprehensive proteomes are necessary to provide a molecular framework to integrate this information. The Golgi complex is the central organelle of the secretory pathway and functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. The methods described in this chapter facilitate the isolation of an enriched stacked Golgi fraction (GF) from rat liver (1,2) and the shotgun proteomic analysis of the fraction using multidimensional protein identification technology (MudPIT) (3,4).