We have developed a new micro-assay for lymphotoxin utilizing murine l-929 target cells prelabeled with technetium-99m, a high specific activity, metastable gamma emitter which is bound to living cells and not released in appreciable quantities by dead or injured cells. Target l-cells prelabeled with 99mTc were added to micro tissue culture plates at a concentration of 10,000 cells/well. Eighteen hr later lymphotoxin or control material was added and the cells were incubated for an additional 12–28 hr. A maximum cytocidal effect was observed after incubation of target cells for 28 hr with 100 CU of lymphotoxin. The number of surviving adherent cells as determined by gamma counting for 99mTc was in accord with that recorded by visual enumeration. The 99mTc microassay is a sensitive, easily performed, rapid test for lymphotoxin activity which appears to have a number of advantages over previously reported in vitro methods. We thank Mrs. L. Williams and S. Vaishnav for technical assistance and Mrs. K. Phipps and E. Olson for secretarial help. © 1973, SAGE Publications. All rights reserved.