An investigation was conducted to evaluate the feasibility of site-selective addition of diagnostic and therapeutic agents to monoclonal antibody F(ab')2 fragments through cross-linking of antibody Fab' fragments. In the investigation, trifunctional equilibrium transfer alkylation cross-link (ETAC) reagents, 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid, 1a, N-[4-[2,2bis[(p-tolylsulfonyl)methyl]acetyl]-benzoyl]-4- (tri-n-butylstannyl)phenethylamine, 3a, and N-[4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]- benzoyl]-4-[125,131I]iodophenethylamine, 3b, were synthesized. The ETAC derivatives were reacted with Fab' fragments of an antirenal cell carcinoma antibody (A6H) produced from reduction of F(ab')2 using 1,4-dithiothreitol. Cross-linking of Fab' was obtained to yield a radioiodinated modified F(ab')2, [mF(ab')2], fragment. The cross-linking reaction produced mixed addition products, requiring the desired mF(ab')2 to be separated from radioiodinated Fab' by size exclusion HPLC. Tumor cell binding immunoreactivities varied (60-90%) for five isolated mF(ab')2 preparations but were consistent with other radiolabeled antibody preparations tested on the same day. In vitro stability testing indicated that the mF(ab')2 was reasonably stable toward loss of the ETAC cross-linking reagent, except under strongly basic conditions. Under reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, protein bands believed to be cross-linked heavy chain dimers were observed. Biodistribution of purified radioiodinated A6H mF(ab')2 was conducted in athymic mice bearing a renal cell carcinoma xenograft (TK-82). A nonmodified control A6H F(ab')2, radioiodinated as a p-[125,131I]-iodobenzoyl conjugate, was coinjected for comparison. The radioiodionated mF(ab')2 had a similar distribution to the radioiodinated control at 3.5, 19, and 43 h postinjection. In another study, the distribution of radioiodinated A6H Fab' was evaluated at 4 and 24 h to establish clearance and pharmacokinetics for comparison with the data obtained from the mF(ab')2. The biodistribution data indicated that A6H mF(ab')2 was quite different from that of A6H Fab'. The results from this preliminary study suggest that it may be possible to attach (large polymeric) diagnostic or therapeutic agents to monoclonal antibody F(ab')2 fragments through the use of ETAC reagents.