The breast and ovarian cancer predisposition gene product BRCA1, binds to BARD1 at its N terminus. In cells BRCA1 is found as a heterodimer with BARD1 and may represent the functionally active form of BRCA1. Using yeast two-hybrid and split-hybrid screens we have identified 16 independent missense mutations which prevent the ability of the BARD1 N terminus to heterodimerize with BRCA1. With reference to the recent structure of the BARD1center dotBRCA1 RING complex (Brzovic, P. S., Rajagopal, P., Hoyt, D. W., King, M-C., and Klevit, R. E. (2001) Nat. Struct. Biol. 8, 833--837) we note two classes of mutation; those that map to the hydrophobic core forming the BARD1:BRCA1 interface and are substitutions of leucine, and those that map to residues forming intramolecular contacts either in helical packing, or in the conserved zinc chelating cysteine residues of the RING itself. The directed mutation of charged residues predicted to play a role in the interaction could not alone prevent heterodimer formation suggesting that, while polar interactions may participate in the specificity of the interaction, they are not crucial. Together these data provide functional evidence for the requirement of a hydrophobic interface and illustrate that disruption of the tertiary structure by mutations away from the interface itself are able to prevent formation of the heterodimer.