Uptake of [2-ring-14C]misonidazole and [3H]misonidazole with tritium in the side chain has been compared in 1-mm EMT-6/UW spheroids using liquid scintillation counting and autoradiography. The uptake of both labeled sensitizers as a function of incubation time was virtually identical. Uptake by the spheroids exceeded levels in the medium by 11/2 to 2 hr and was well modeled as a first-order binding process, with rate constants of 0.00324 hr-1 for 3H and 0.00388 hr-1 for 14C. The similar uptake of the two versions of this sensitizer labeled in different positions suggests that the metabolic actions which allow the drug to bind in hypoxic cells do not principally involve metabolites which separate the number 2 carbon of the imidazole ring from the side chain. The pattern of silver grains in autoradiographs was similar for both labeled sensitizers, with most labeled drug bound in an intermediate zone of cells between the necrotic center and the actively proliferating rim of the spheroids. The superior resolution possible with the tritiated compound showed that both nucleus and cytoplasm in viable looking cells were labeled while pycnotic cells were not labeled.