Although intact monoclonal antibodies (MAbs) are well suited as therapeutic reagents, their relatively slow clearance rates render them less useful for imaging applications. Over the last several years, our laboratory has developed a unique targeting approach to solid tumors that utilizes MAbs directed against DNA and its components to bind to degenerating cells and necrotic regions of tumors in a specific manner. Because these MAbs have considerable potential for the early diagnosis of cancer and for the monitoring of cytoreductive therapies, the availability of an effective imaging agent is highly desirable. To accomplish this goal, a series of genetically engineered derivatives of MAb chTNT-3 including the single-chain Fv, diabody, triabody, Fab, and F(ab')(2) were generated and expressed in NS0 myeloma cells using the Glutamine Synthetase Amplification System. Initial in vitro studies demonstrated that each of the antibody derivatives maintained its antigen binding in a stable manner. In vivo analyses after radiolabeling were then performed to evaluate their pharmacokinetic, biodistribution, and tumor-imaging properties in solid tumor-bearing mice. The results of these studies showed that compared with intact parental chTNT-3, which has a half-life of 134.2 h, the smaller derivatives were eliminated more rapidly (4.9-8.1 h). Importantly, the smaller derivatives were found to have significantly higher tumor-to-organ ratios, but lower overall uptake levels compared with parental (125)I-chTNT-3 in two different tumor models. A comparison of the five derivatives showed that the F(ab')(2) reagent consistently gave the best results in imaging and biodistribution studies. Based upon these results, further studies are warranted to demonstrate the potential of this reagent for the diagnosis and monitoring of solid tumors using noninvasive imaging techniques such as immunoscintigraphy and positron emission tomography (PET).