Radiolabeled murine monoclonal antibody TNT-1, directed against the nuclear histones of degenerating cells, was used to treat human colon adenocarcinoma HT-29 spheroids in vitro. The therapeutic effects of 131I-TNT-1 were investigated as a function of the radioactive dose, treatment time, and number of treatments. Efficacy of treatment was assessed by TNT-1 antibody uptake, spheroid growth delay, and morphological examination using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). From these studies, it was determined that the therapeutic effect increased with the number of doses and the duration of treatment. Spheroids treated for 24 h showed approximately two to four times more cell death than those with a 2-h treatment. As previously shown in animal models, additional treatment with radiolabeled TNT-1 produced an expanding number of TNT-1 targets, and subsequent treatments were more effective as shown by antibody uptake studies. Microscopic examinations demonstrated that morphological changes consistent with spheroid destruction correlated well with antibody uptake data and increased gradually with dose, treatment time, and frequency of treatments. At the ultrastructural level, destruction of cells in the treated spheroids included the formation of porous cell membranes, crater-like holes (SEM), blebbing, and dissolution of cytoplasmic organelles (TEM). With continued culture, the injured spheroids were found to disaggregate after intensive 131I-TNT-1 therapy (e.g. 50 microCi/ml or 100 microCi/ml with two or three 24-h treatments). These findings suggest that tumor spheroids can be used as an in vitro model to evaluate monoclonal antibody therapy using TNT-1 and other candidate mAbs directed against intracellular antigens exposed in degenerating cells of tumors.