To develop a suitable vector and an administration technique for in vivo gene transfer, the tissue distribution of plasmid DNA (pDNA) needs to be understood. In this study, a novel residualizing radiolabel for pDNA was developed. 4-[p-Azidosalicylamido]butylamine (ASBA) was coupled with diethylenetriaminepentaacetic acid (DTPA) anhydride, then the conjugate was reacted with pDNA by photoactivation, followed by labeling with [(111)In]InCl(3) to obtain (111)In-pDNA. The overall structure of pDNA was well preserved, and the retention of its transcriptional activity was 40-98%. After intravenous injection of (111)In-pDNA into mice, about 50% of the radioactivity was recovered in the liver within 3 min. The level remained stable for at least 2 h, followed by a very slow decrease to 45% at 24 h. This contrasted with the results obtained with (32)P-pDNA by nick translation, in which a rapid decrease in hepatic radioactivity was observed. The amount of radioactivity in the lung following the administration of polyethyleneimine/(111)In-pDNA complexes correlates well with the transgene expression. These results indicate that the novel residualizing radiolabel clearly demonstrates the cells that have taken up pDNA and, therefore, gives us useful information about how to design a better approach for nonviral in vivo gene delivery.