Determining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution. Display omitted ► DPiM provides a large-scale map of metazoan protein complexes ► DPiM defines 556 protein complexes and assigns novel members to several of them ► The map provides functional links and biological context to uncharacterized proteins ► DPiM defines a resource for the analysis of protein complex evolution A comprehensive map of protein interactions in a metazoan provides functional information about uncharacterized proteins and yields insight into the evolving intricacy of protein complexes.