Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.