Cortisol 3-(O-carboxymethyl)oxime, 6- and 21-hemisuccinoxycortisol, and cortisol 7-carboxyethyl thioether were synthesized. These carboxyl derivatives were labeled using a described general labeling method with an europium chelate. The labeled steroids were tested in a competitive time-resolved fluoroimmunoassay using antibodies raised against cortisol. Only the site-homologous antigen-antibody pairs underwent immunoreaction and gave satisfactory calibration curves.