Cross-linked F(ab')2 fragments derived from PR1A3, a murine monoclonal antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as follows: Digestion of PR1A3 with pepsin gave F(ab')2 fragments which were purified by ion-exchange chromatography. Fab' was produced by reduction of F(ab')2 with cysteine. Following reaction with BMH, cross-linked F(ab')2 fragments, XL-F(ab')2, were isolated by preparative size-exclusion HPLC. Analysis by HPLC and SDS-PAGE demonstrated the presence of a molecule of approximately 100 kDa containing a nonreducible 50,000 MWt chain. Competitive and direct radioligand binding assays demonstrated that the XL-F(ab')2 had a capacity to bind to antigen similar to that of unmodified F(ab')2. The biodistribution of 125I-labeled XL-F(ab')2 and unmodified F(ab')2 was compared in a nude mouse human tumor xenograft model at 4, 24, and 48 h after injection. Differences between the two preparations were most significant after 24 or 48 h. Tumor uptake of the XL-F(ab')2 was greater and normal tissue retention less than with the unmodified fragment. Tumor to normal tissue ratios at 48 h ranged from 6.2 to 35.2 for XL-F(ab')2 while for the normal F(ab')2 they ranged from 1.5 to 14.2. These results suggest that cross-linked antibody fragments may produce better tumor targeting in clinical application.