In previous studies, we have compared the immunochemical properties, the in vivo pharmacokinetics, and the tumor penetrance of a radioiodinated single-chain Fv (sFv) in comparison with other immunoglobulin (Ig) forms (intact IgG, F(ab′)2, and Fab′) (Cancer Res., 51: 6363–6371, 1991). Biodistribution studies demonstrated a higher percent injected dose/g in the liver and spleen for the intact IgG and F(ab′)2. Renal uptake was observed with the Fab′ and F(ab′)2, whereas the sFv demonstrated no specific localization in either of these organs. The 125I-labeled sFv also demonstrated a more even distribution throughout the tumor xenografts as compared to the other Ig forms (Cancer Res., 52: 3402–3408, 1992). Subsequent studies utilizing the sFv conjugated with a radiometal (177Lu) demonstrated that the sFv was being metabolized by the kidney, and a significantly higher percent injected dose/g was obtained with a 177Lu-labeled sFv as compared to a 125I-labeled sFv (Cancer Res., 52: 6413–6417, 1992). These previous studies indicated the potential utility of radioiodinated sFv and other Ig fragments for use in radioimmunoguided surgery with a hand-held probe, diagnostic imaging, and possibly therapy. The present study compares the distribution in normal tissues of the 4 Ig forms of monoclonal antibody (MAb) CC49, which is directed against a pancarcinoma antigen (tumor-associated glycoprotein-72). 125I-labeled sFv, Fab′, F(ab′)2, and IgG of MAb CC49 were administered to athymic mice either bearing or not bearing the tumor-associated glycoprotein-72 positive human colon carcinoma xenograft (LS-174T). At various intervals following the i.v. injection of the Ig forms, the liver, spleen, kidneys, and lungs were removed for autoradiographic analyses. Dramatic differences were observed in the kidney; the IgG was found only in the renal vasculature, whereas the Fab′, F(ab′)2, and sFv showed a high density of grains in the cortical tubules. In the liver, the IgG and F(ab′)2 were found in association with hepatocytes, Kupffer cells, and in the sinusoids; the Fab′ and sFv were primarily associated with the Kupffer cells. In the spleen, the Ig forms localized to the marginal zones surrounding the lymphoid follicles. No specific accumulation of grains for any of the Ig forms was observed in the lung. In each of the tissues, the clearance rates were related to the size of the Ig form. The localization in the liver and spleen was determined to be antigen-mediated. No specific localization was observed when 125I-labeled BL-3, an isotype-matched control MAb, was injected into tumor-bearing mice, or, when the 125I-labeled CC49 IgG was administered to non-tumor-bearing mice. By defining the interactions of a MAb and its modified forms not only with tumor lesions but also with normal organs and tissues, more rational protocols for uses of a MAb and its various forms, including sFv's, can be designed for diagnostic and therapeutic applications.