The two-dimensional crystallization of the protein streptavidin under biotinylated lipids has provided a convenient model system for studying the molecular mechanisms underlying protein crystallization: At neutral pH, streptavidin forms crystals with C222 symmetry and an X-shaped morphology that arises from asymmetric growth rates along perpendicular axes. The presence or absence of biotin association in the subunits has been connected to the growth asymmetry, but the molecular mechanism coupling biotin binding and growth kinetics has remained uncertain. Here we show evidence that a cooperative interaction between the Asn 23 hydrogen bond to biotin and a protein-protein hydrogen-bonding interaction at Tyr 22-Thr 20 across the crystal contact interface is acting as the structural/energetic connection between biotin binding and the asymmetric crystal growth kinetics. Brewster's angle microscopy and atomic force microscopy studies of a Thr20Ala site-directed mutant revealed that it crystallizes as symmetric squares, while maintaining the molecular C222 arrangement of wild-type protein crystals. These results suggest that the T20-Y22 hydrogen-bond interaction across the protein-protein contact interface is the crystallization contact that is altered by biotin binding to give growth asymmetry in wild-type crystals.