As part of a search for optimal conditions for radioimmunotherapy of lymphoma, rituximab was labeled with 2 different specific activities of 131I and immunoreactivity was comparatively measured. METHODS: Labeling was performed with chloramine T using as starting conditions 185 MBq of 131I per 1 mg and per 5 mg of antibody for labelings A and B, respectively. Six comparative labelings were performed over a period of 10 mo with similar efficacy and purified by anion-exchange chromatography. Immunoreactivity was determined immediately after labeling in parallel assays using different concentrations of fresh Raji and Daudi cells. Results were compared at maximal observed specific binding on 10(7) cells and after extrapolation to infinite antigen excess. A statistical analysis was performed to predict the frequency of radiolabeled mono- and polyiodinated antibodies: First, a gaussian distribution predicted the number of iodine atoms per antibody in labelings A and B, respectively; then, the radiolabeling probability was developed according to the Newton binome. RESULTS: Final radiochemical purity was >98.4% for all labelings. The final mean specific activities were 169.7 MBq/mg and 32.8 MBq/mg, corresponding to 0.87 and 0.17 iodine atoms per antibody in labelings A and B, respectively. Labeling B showed a significantly higher immunoreactivity than did labeling A, the mean relative increase in binding being > or =28% for both Raji cells and Daudi cells. The predictive statistical analysis indicated that 57.3% and 15.4% of radiolabeled antibodies in labelings A and B, respectively, were polyiodinated. CONCLUSION: The low specific activity of 131I-rituximab allowed preservation of a high immunoreactivity and correlated with the prediction of a low percentage of polyiodinated radiolabeled antibodies.