Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized. Five clones are IgG(1), two clones are IgM and one clone is IgG(2b); all have kappa light chain. The affinities are in the range of 1.1 x 10(-7) approximately 2.4 x 10(-9) M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal. Each clone was constructed as single-chain Fv (scFv) and expression was performed in E. coli. Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The nucleotide and amino acid sequences of V(H) and V(L) of four scFvs were deduced and their family and subgroup were analyzed. We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct. The E. coli-expressed scFvs showed 1.5 x 3.4-fold lower affinities (2.8 x 10(-8) approximately 3.6 x 10(-9) M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited approximately 10(-6) M of affinity.