We compared three different methods for radioiodination of human spleen ferritin. Tracers prepared by direct oxidative iodination with use of Chloramine T or 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodogen) were structurally altered by the labeling procedure, as was made apparent by gel filtration and electrophoresis on polyacrylamide gel. Tracers prepared by conjugation to radioiodinated N-succinimidyl-6-(4-hydroxyphenyl)propionate retained their structural integrity, as assessed by physicochemical methods. Such tracers bound more than 90% in antibody excess, yielded self-displacement curves parallel to unlabeled ferritin standards, and retained their immunoreactivity for nine weeks. By contrast, immunological activity and stability of tracers prepared by the oxidative methods were less satisfactory. Tracers prepared by using lodogen were unsuitable for use in the ferritin radioimmunoassay because of shallow standard-inhibition curves and poor binding in the absence of added standard. Thus, we found the conjugation procedure to be the most suitable of the three methods for preparation of spleen-ferritin tracers.