The two-dimensional crystallization of streptavidin at biotinylated interfaces provides a model system for elucidating the role of interfacial binding dynamics in determining crystal morphologies and phases. In this study, a library of eight well-characterized site-directed mutants with increased biotin dissociation rates has been compared to core streptavidin (dissociation half-life of 59 h). The W79F, W120F, Y43F, and S27A mutants, with half-lives of 9.8 h, 38 min, 20 min, and 10 min, respectively, all displayed the "X"-shaped crystal morphology that is characteristic of core streptavidin. A sharp change in morphology is observed with the N23E (7.2 min) and N23A (4.8 min) mutants. The N23E mutant crystallizes in rectangular shapes, and the N23A displays square crystal morphology. The D128A mutant (1.7 min) crystallizes in elongated needles, but this is the one mutant which displays significant three-dimensional structural alterations. The W120A (<1 min) mutant did not display significant interfacial binding and did not crystallize. Quantitative Brewster angle microscopy was used to characterize the crystallization process. The noncrystalline background remained in equilibrium with the crystalline regions, and thus all the mutants crystallized as a first-order phase transition. The critical surface concentration for crystallization remained constant until the half-life reached 10.4 min for S27A. The square-shaped N23A crystal displayed the lowest critical surface concentration, which was equivalent to the values observed previously for square-shaped crystals obtained via metal binding. Fourier analysis of transmission electron microscopy images demonstrated that all of the mutants crystallized with the same C-222 space group characteristic of core streptavidin. The change in crystal shape over a sharp range of dissociation rates is consistent with a change in a rate-limiting microscopic kinetic step that underlies macroscopic morphology. Alternatively, the altered shapes observed with the Asn 23 mutants could be the result of changing structural and energetic coupling between biotin binding and the directly adjacent Thr 20 and Tyr 22 residues which hydrogen bond to each other across the protein-protein crystal contacts.