We have cloned and sequenced the Fd and kappa chains of a monoclonal antibody against 17β-estradiol is order to study the binding site at the molecular level. The chains were cloned into the vector pComb3 which was used both for Fab production and for phage display in E. coli. The cloning sites in the vector are designed for the in-frame fusion of the amino termini of the mature Fd and light chains to pelB signal sequences, to enable secretion to periplasm where Fab assembly occurs.The integrity of the recombinant Fab was assessed by comparison of its binding characteristics to a proteolytic Fab fragment of the parental hybridoma IgG. The affinity for estradiol was 2-fold decreased and cross-reactivity to testosterone was 3.5-fold increased. Since the cloning sites for the Fd and the light chain in the pComb3 vector were embedded in the framework 1 coding sequence, the amino termini of the chains were not strictly authentic. Keeping this as a tentative explanation for the observed changes in steroid binding, we removed the embedded cloning sites to restore the original coding sequences. The revised Fab showed binding properties identical with the proteolytic fragment, confirming that the observed differences resulted from changes in the amino termini. Furthermore, restoration of the heavy chain alone affected for estradiol while the restoration of the heavy chain alone affected only testosterone cross-reactivity. The restoration of the N-Terminal sequences also improved the Fab yield and immunoreactivity by a factor of 3, primarily attributed to the influence of the heavy chain N-terminus.We have shown that the very first N-terminal residues of the antibody are able to modify hapten binding, despite the fact that they cannot be in direct contact with the cleft-bound hapten.