Anti-idiotypic (Anti-Id) monoclonal antibodies (MoAbs) against anti-carcinoembryonic antigen (CEA) monoclonal antibody 5B3 (IgG1) were prepared and characterized. Five anti-Id MoAbs recognized the private idiotype of MoAb 5B3. Idiotype mapping suggested that MoAb 5B3 had at least three distinct idiotopes at its antigen combining site. Utilizing the inhibition of idiotype-anti-idiotype interaction with use of anti-Id MoAb T4-305 or T4-212, an immunoassay to detect shedding CEA was established. The results of this inhibition assay correlated with those of double determinant immunoassay and inhibition percentages linearly increased in a CEA concentration-dependent manner. Since it requires only one epitope of a given antigen molecule, this assay could be widely used for detection of shedding antigens, including tumor-associated antigens.