For the therapy of cancer patients whose disease is positive for Carcinoembryonic Antigen (CEA), we developed an active specific immunotherapy based on the idiotypic network. The anti-idiotype monoclonal antibody (mAb), 3H1 was generated by immunization of mice with the anti-CEA mAb, 8019. 3H1 mimics CEA both functionally and structurally and acts as a surrogate for CEA. To define the minimum structural requirements for antigen mimicry by 3H1, we constructed plasmid vectors for expression of single chain Fv (scFv) variants of 3H1 in Escherichia coli. Variable heavy (VH) and variable light (VL) chain domains of 3H1 were linked by a 15 amino acid linker (Ln), (Gly4Ser)3 in two constructs, VH-Ln-VL and VL-LnVH. Ln was omitted in two constructs, VH-VL and VL-VH. Each of the scFv constructs has a tag of six His [(His)6 tag] for purification by metal chelate affinity chromatography and detection by enzyme-linked immunoabsorbent assay (ELISA). Comparisons of the binding of 8019 to purified scFv proteins by ELISA and immunoblot experiments showed that only VH-Ln-VL had significant activity. VH-Ln-VL also showed maximum inhibition of binding of 8019 to CEA. Immunization of mice with naked VH-Ln-VL and VH-Ln-VL conjugated to keyhole limpet hemocyanin induced anti-CEA antibodies in mouse sera. Sera from immunized mice inhibited the binding of 8019 to 3H1 as well as CEA. Induction of anti-CEA antibodies in the immunized mice was confirmed by flow cytometric analysis using CEA positive MC-38cea cells. These results demonstrate that for antigen mimicry of 3H1 scFv, the presence of Ln is necessary and the domain order should be VH followed by VL.