The existence of interactions between many cellular proteins and various polyanionic surfaces within a cell is now well established. The functional role of such interactions, however, remains to be clearly defined. The existence of protein arrays, with a large selection of different kinds of proteins, provides a way to better address a number of aspects of this question. We have therefore investigated the interaction between five cellular polyanions (actin, tubulin, heparin, heparan sulfate, and DNA) and ϳ5,000 human proteins using protein microarrays in an attempt to better understand the functional nature of such interaction(s). We demonstrate that a large number of polyanion-binding proteins exist that contain multiple positively charged regions, are often disordered, are involved in phosphorylation processes, and appear to play a role in protein-protein interaction networks. Considering the crowded nature of cellular interiors, we propose that polyanion-binding proteins interact with a wide variety of polyanionic surfaces in cells in a functionally significant manner.Our oldest view of cells as lipid bags filled with proteins and nucleic acids long ago gave way to a much more complex picture in which interior membranes and fibrous proteins create a large variety of unique environments that are critical for individual cellular functions. This picture continues to be refined, but the idea that lipid membranes are the most important structural components defining local environments continues. It is now established that many cellular proteins are capable of interacting with a wide variety of polyanions. We have recently suggested that both the interior and exterior polyanionic surfaces may also have an important role to play in the organization of cellular function. If one views the typically very crowded interior of a cell filled with microtubules, microfilament, ribosomes, lipid membranes, and other structures (1) in the light of the polyanionic nature of these molecular entities (2), the interior of cells can be seen as a highly polyanionic environment offering an extensive potential interaction surface. Most of our knowledge about polyanion-protein interactions stems from in vitro studies that have traditionally been conducted at low concentrations. Under these conditions, the effects of macromolecular crowding and the presence of polyanionic surfaces are neglected. The concept of macromolecular crowding refers to the presence of cell organelles, proteins, polysaccharides, cytoskeleton proteins, and membranes that produces severe steric confinement and a resultant high thermodynamic activity of the molecules present (3, 4). When considering the large surface area and the importance of cellular polyanions such as actin, tubulin, and ribosomes in a crowded cellular environment, the nonspecific electrostatic interaction of proteins containing positively charged regions with these polyanions can be expected. In such cellular matrices, biochemical reaction rates, equilibria, and the diffusion of macromolecules as...