Collective motion, where large numbers of individuals move synchronously together, is achieved when individuals adopt interaction rules that determine how they respond to their neighbors' movements and positions. These rules determine how group-living animals move, make decisions, and transmit information between individuals. Nonetheless, few studies have explicitly determined these interaction rules in moving groups, and very little is known about the interaction rules of fish. Here, we identify three key rules for the social interactions of mosquitofish (Gambusia holbrooki): (i) Attraction forces are important in maintaining group cohesion, while we find only weak evidence that fish align with their neighbor's orientation; (ii) repulsion is mediated principally by changes in speed; (iii) although the positions and directions of all shoal members are highly correlated, individuals only respond to their single nearest neighbor. The last two of these rules are different from the classical models of collective animal motion, raising new questions about how fish and other animals self-organize on the move.collective animal behavior | fish shoals | group motion | self-propelled particles | self-organization C ollective motion of animal groups occurs when multiple individuals move synchronously, producing large scale "flocking" patterns (1-5). Numerous models have been developed to describe patterns of collective motion in terms of interactions between individuals (6-9). These simulation models usually assume that individuals move at a constant speed and their interactions are mediated through direction changes (1). Often these models use zonal rules, where individuals move away from neighbors at close distances and align and/or move toward neighbors at greater distances. Interactions can be with either all neighbors within some zone (7) or with a set of n nearest neighbors (10). These and other models have succeeded in reproducing qualitatively similar patterns to those seen in the collective motion of animal groups in nature.It remains unclear, however, whether the interaction rules implemented in models are the ones used by animals. Indeed, many collective motion patterns observed in nature can be simulated by models using very different interaction rules (1). We are only now beginning to accumulate evidence about which interaction rules are used. There has been recent identification of zones of repulsion and alignment in surf scoters (11). The structure of starling flocks is consistent with topological interactions between the birds (10). Homing pigeons appear to have hierarchical interactions such that birds with higher route-following fidelity act as leaders (12)(13)(14)(15). Partridge showed that lateral line and vision are both important in producing directional alignment in Gadid fish (16). Nonetheless, there remain a large number of open questions about the interactions of fish. For example, do fish adopt attraction and alignment within distinct zones as purported in most models? How many neighbors do fish ...