Abstract: Protein interactions regulate the systems-level behavior of cells, thus, deciphering the structure and dynamics of protein interaction networks in their cellular context is a central goal in biology. We have performed a genome-wide in vivo screen for protein-protein interactions (PPIs) in Saccharomyces cerevisiae by means of a protein-fragment complementation assay (PCA). We identified 2,770 interactions among 1,124 endogenously expressed proteins. Comparison with previous studies confirms known interactions, but most are new, revealing a previously unknown sub-space of the yeast protein interactome. PCA detects structural and topological relationships between proteins, providing an 8-nanometer resolution map of dynamically interacting complexes in vivo and extended networks that provide insights into fundamental cellular processes, including cell polarization and autophagy, pathways that are evolutionarily conserved and central to both development and human health.