Hydroxylapatite high performance liquid chromatography was used to prepare two fractions from 125I- Fab 96.5. One fraction (peak 1) had relatively low immunoreactivity (25-38%) and the second fraction (peak 2) had high immunoreactivity (70-81%). Scatchard analysis showed similar affinity constants for the two preparations (2.9 x 10(9) M-1 for peak 1; 3.4 x 10(9) M-1 for peak 2). In biodistribution and imaging studies in athymic mice with human melanoma (FEMX-II) xenografts the high immunoreactivity preparation rapidly cleared from the blood and nontumor organs while retention of radioactivity in the tumor was prolonged. The low immunoreactivity preparation, had slower blood and nontumor organ clearance but faster tumor clearance than the high immunoreactivity fraction. Thus, in these studies highly immunoreactive antibody gave higher tumor to nontumor ratios and enhanced the target to nontarget image contrast.