Molecular interactions for proline in a highly concentrated aqueous solution (up to 1:5 proline:water molecular ratio) have been investigated using a variety of experimental and computational techniques. Rather than the solution containing either small crystallites or large aggregates of proline, three-dimensional structural analysis reveals the presence of prolineproline dimers. These dimers appear to be formed by cyclic electrostatic interactions between CO2 and NH2+ groups on neighboring proline molecules, which causes the ring motifs of proline to be roughly parallel to one another. In addition, water appears to aggregate around the electrostatic groups of the prolineproline dimers where it may in fact bridge these groups on different molecules. The observed short-range interactions for proline in solution may explain its function as a hydrotrope in vivo in which this observed dimerization might allow proline molecules to generate small pockets of a hydrophobic environment that can associate with nonpolar motifs of other molecules in solution. The results presented here emphasize the need for careful three-dimensional analysis to assess the short-range order of highly concentrated solutions.