The crystal structure of a murine mAb, 4C3, that binds to the C-terminal lobe of the cockroach allergen Bla g 2 has been solved at 1.8 Å resolution. Binding of 4C3 involves different types of molecular interactions with its epitope compared with those with the mAb 7C11, which binds to the N-terminal lobe of Bla g 2. We found that the 4C3 surface epitope on Bla g 2 includes a carbohydrate moiety attached to Asn(268) and that a large number of Ag-Ab contacts are mediated by water molecules and ions, most likely zinc. Ab binding experiments conducted with an enzymatically deglycosylated Bla g 2 and a N268Q mutant showed that the carbohydrate contributes, without being essential, to the Bla g 2-4C3 mAb interaction. Inhibition of IgE Ab binding by the mAb 4C3 shows a correlation of the structurally defined epitope with reactivity with human IgE. Site-directed mutagenesis of the 4C3 mAb epitope confirmed that the amino acids Lys(251), Glu(233), and Ile(199) are important for the recognition of Bla g 2 by the 4C3 mAb. The results show the relevance of x-ray crystallographic studies of allergen-Ab complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.