Two new antibody forms of the general structure F(ab')n (n = 3 or 4) were prepared and tested in vivo as part of an ongoing search for antibody candidates with improved biodistribution properties for cancer immunotargeting applications. The novel multivalent antibody forms, called F(ab')3-x (tribody, 150 kDa, x = cross-linker) and F(ab')4-x (tetrabody, 200 kDa), were constructed through chemical cross-linking of Fab' subunits derived from murine CC49 IgG, a monoclonal antibody which recognizes the tumor-associated antigen TAG-72. Two new chemical reagents (trismaleimide 1 and tetramaleimide 2) were synthesized for use in cross-linking cysteine sulfhydryl groups present on the hinge region of Fab'. Homogeneous Fab' was prepared by mild reduction of F(ab')2 followed by selective reoxidation of interchain disulfide bonds, leaving a single hinge-region cysteine sulfhydryl group available for modification. For biodistribution studies, the parent F(ab')2 fragment was first radiolabeled via lysine amine modification using the isothiocyanate derivative of the 105Rh(BA-2,3,2-tet)Cl2 complex. Both new fragment forms were shown to retain antigen binding ability in vitro using a solid-phase immunoassay. Although isolated yields for F(ab')3-x and F(ab')4-x were low (18 and 4%, respectively), sufficient quantities were prepared for preliminary biodistribution studies in Balb/c mice and, in the case of F(ab')3-x, for a 5-day biodistribution study in tumor-bearing nude mice. A large proportion of the 105Rh-labeled F(ab')4-x was found to accumulate in the liver, possibly indicating an upper size limit for the in vivo use of cross-linked fragments. The biodistribution behavior of 105Rh-labeled F(ab')3-x in both Balb/c and nude mice was intermediate between that of IgG and F(ab')2 for all organs studied. Kidney localization was reduced, while blood circulation time and tumor accumulation were slightly increased, for the trivalent species compared with F(ab')2. The unique biodistribution profile of F(ab')3-x suggests the possible use of this multivalent fragment for in vivo tumor targeting applications.