Bacteria are often thought to be the very simplest organisms, but this view is being quickly replaced by an appreciation for the very complex behavior and social lives of bacteria. We now know that most bacteria in nature exist in massive, three-dimensional communities called biofilms, which are often likened to 'microbial cities'. Others even suggest that biofilms are most similar to multicellular tissues, like those that make up plants and animals. In either case, we now know that bacteria communicate through the use of chemical signals, migrate in concert to build multicellular structures, and pay a metabolic 'cost' to produce an array of shared, beneficial molecules. This last form of cooperation seems especially challenging to explain: why would evolution favor a cooperative type, if a selfish type can get all of the same benefits without paying a cost? There could be three possible answers to this question: 1) perhaps a cell that produces a beneficial molecule can itself benefit more than a 'free rider' neighbor who does not, 2) because bacterial reproduction is clonal, 'neighbors' in biofilms tend to be clone-mates, which means cooperators will tend to be around cooperators while free riders are near free riders, or 3) entire biofilms with more cooperators will contribute more offspring to the global population than biofilms with more free riders. The proposed experiments will precisely measure competition between cooperators and free riders in the human pathogen Pseudomonas aeruginosa using three-dimensional fluorescent microscopy to map the spread of each type in a growing biofilm. These experiments will strengthen an established, interdisciplinary collaboration between William Driscoll and the geneticists Leland and Elizabeth Pierson and greatly enhance the training of William Driscoll and an undergraduate assistant in methods in microbiology and confocal microscopy. Finally, the results of these important experiments will be presented at the international scientific conference Evolution in 2011 and published in a peer-reviewed scientific journal.