AbstractThe antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen‐binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen‐specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour‐associated antigen mucin‐1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen‐specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity. Copyright © 2004 John Wiley & Sons, Ltd.