We have developed a radioisotopic microassay of cell-mediated immunity employing target cells prelabeled with technetium-99m (99mTc), a high specific activity metastable gamma emitter. Labeling kinetics, release and reutilization, subcellular localization, and effects of 99mTc on DNA and protein synthesis have been investigated. Target cells were optimally labeled with 10 mCi of 99mTc at 37 °C for 10 min. Cyclic freezing and thawing released less than 10% of total bound radioisotope. Spontaneous leakage of 99mTc by monolayer cells was negligible over 48 hr and that which was released appeared to be nonreutilizable. Cell fractionation revealed that nuclear, mitochondrial, and microsomal fractions all were labeled with 99mTc. The incorporation of 3H-thymidine and 3H-amino acids was not impaired in 99mTc-labeled cells. The alloimmune reactivity of C57BL/6 mice which had received A/J skin allografts was studied by means of the 99mTc microcytotoxicity assay. Cell-mediated immunity was clearly evident at 7 days postgrafting, peaked at 14 days, and had declined to background levels by 21 days. These findings correlated well with initial acceptance and ultimate rejection of the allografts. The rapid labeling time without dependence upon cell division for incorporation, high specific activity, low spontaneous release, and nonreutilizability are important advantages of 99mTc over other radionuclides which have been employed in in vitro assays of cell-mediated immunity. © 1974.