For the development of calixarene-based radiotherapeutic agents, the conjugation to biomolecules and immunogenicity in mice of potential 225Ac3+-chelating calixarenes were studied. A calixarene triethyl ester isothiocyanate and a bis(calixarene) hexacarboxylic acid, containing a masked thiol functionality, were used in conjugation experiments to a mouse monoclonal antibody and serum albumins. All characterization techniques indicate that only the calixarene carboxylic acid is successfully conjugated to the biomolecules. The immunoreactivity of the conjugates is not impaired when up to 6 equiv of calixarene are bound to the monoclonal antibody. Animal tests indicated that the immunogenicity toward the calixarene is strongly influenced by the nature of the carrier, the dosage, and the injection method. No immune response occurred when a homologous carrier was used or when a heterologous carrier was applied at a dosage of 10 μg per immunization via intravenous injection. Under all other conditions, the presence of antibodies directed against the calixarene was demonstrated. Thus, for the application in radioimmunotherapy, the conjugation of a calixarene to a humanized antibody will probably not lead to an immune response, and the immunoreactivity will not be disturbed.