The generation of anti‐IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (FcER) interaction site has been determined using cross‐inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the FcεR interaction site and the binding sites of various anti‐IgE mAb, we employed site‐directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant E heavy chain (Cε) gene bearing a (4‐hydroxy‐3‐nitrophenyl)acetic acid (NP)‐binding VH region. Several site‐specific mutants in the Cε3 and Cε4 domains of this recombinant Cε gene were prepared and expressed by transfection into the light chain‐producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti‐IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the Cε3 domain, a mutant with a truncated Cε4 domain, a mutant with a 45 amino acid deletion in the carboxy end of Cε3, and a chimeric human Cε in which the human Cε3 was replaced by the homologous mouse Cε3 domain. These mutants have permitted the localization, to the Cε3 domain, of the epitopes recognized by the 84.1C and 95.3 anti‐IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the FcεR binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the FcεR binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE‐FcεR interaction, has been mapped to the Cε4 domain. When tested for binding to the FcεR on RBL‐2H3 cells, the point mutant bound to the FcεR with twofold reduced affinity, while the Cε3 deletion mutant and the mutant truncated in Cε4 lost all receptor binding activity. These data suggest that the FcεR binding site can be assigned to the third C region domain, and that the fourth domain, while not directly involved in FcεR binding, may play a role in the formation of the H2L2 tetrameric IgE molecule, and in stabilizing the conformation of IgE required for FcεR binding.