A cDNA for NCA-50 was cloned into the inducible expression vector pTRB1, using the polylinker site at the C-terminus of the lac Z' gene. An NCA-specific MAb (N1), NCA and CEA cross-reactive MAbs (T84.1, 192) and polyclonal antisera (anti-NCA and anti-CEA, as well as anti-PS beta G) detected the fusion protein, with a mol. wt of 155,000, which constituted about 5% of the total bacterial protein. Deletion and mutation analysis showed that all MAbs which stained positive in western blots mapped to a small region within the last third of the N-terminal domain. Superimposition of the deduced amino acid sequence of NCA-50 on the known structure of immunoglobulins reveals that the antigenic region is located on a surface loop, which corresponds to a fourth hypervariable region on the immunoglobulin heavy chain variable regions. By oligonucleotide directed site-specific mutagenesis amino acids were deduced, which constitute part of an epitope, to which the NCA-50-specific MAb, N1, binds.