The in vivo detection of tumors by immunoscintigraphy with the use of radiolabeled monoclonal antibodies (MoAb) is a new diagnostic procedure currently undergoing clinical evaluation. In the present study the use of 99mtechnetium (99mTc) for this purpose was explored. A simple method for the labeling of microgram quantities of MoAb with 99mTc based on the substitution reaction of MoAb and tetrachloronitridotechnetate ion (99mTcNCl4-) is described. The selective activity of the 99mtechnetium-nitrido-MoAb (99mTcN-MoAb) complexes was proved in vitro by a binding assay with different target cells. The 99mTcN-MoAb complexes were shown to bind reactive cells up to 20 times more avidly than nonreactive cells. The specificity of the 99mTcN-MoAb complexes was shown in vivo. (C57BL/6 X BALB/c)F1 mice bearing palpable tumors (0.3-1.5 cm in diameter) were given an iv injection of 1 of 2 MoAb (one reactive and the other nonreactive) identically labeled with 99mTcNCl4- and then scanned with a gamma camera, and/or the tissues were removed and the localization of 99mTc-nitrido group-labeled MoAb was measured. Tumor localization of the reactive MoAb (1.8-2.2% of the injected dose) was four times greater than that of the nonreactive 99mTcN-MoAb (0.3-0.4% of the injected dose). The localization of specific 99mTcN-MoAb to a murine thymoma was observed in the gamma camera image at just 2 hours after injection. At 27 hours, tumors could readily be detected by 99mTcN-MoAb without the need for background subtraction. Nonreactive 99mTcN-MoAb did not image the tumors. The use of 99mTcN-MoAb offers substantial improvement over radioiodinated (125I or 131I) MoAb for the detection of tumors. The use of 99mTcNCl4- as a labeling agent results in 99mTc-labeled MoAb with high specific activity and specificity when compared with the specific activity and specificity of the 99mTc-MoAb prepared by using the conventional SnCl2 reduction of pertechnetate.