Spheroids of the human prostatic adenocarcinoma cell line DU 145 were used to study experimental radioimmunotherapy. Spheroids were incubated with the 131I-labelled monoclonal E4 antibody until the radionuclide immunoconjugate had bound the 5 to 6 outermost cell layers of the spheroids. A set of 50 spheroids were exposed, either immediately or 48 hr after antibody incubation and washings, to a dilute trypsin solution with the aim of stripping off cells from the spheroid surface. Stripped cells were collected in fractions corresponding to defined spherical shells. Cells were subsequently plated for clonogenic growth. The technique of automated sequential trypsinization of spheroids followed by a clonogenic survival assay permits studies on therapeutic efficacy for radionuclide immunoconjugates on cells from different layers of spheroids. In addition, the absorbed doses throughout a spheroid were calculated. The binding and retention kinetics of the radionuclide immunoconjugate and the excess of 131I-E4 in the culture medium during incubation are factors that were all accounted for in the calculations. If the calculated absorbed doses were inserted into the linear-quadratic survival model and the low dose rate was taken into account, survival values were well in accordance with the experimentally obtained values. The results demonstrate that the 131I-labelled E4 antibody is capable of sterilizing cultured tumour cells that have bound the radionuclide immunoconjugate and, by means of radiation "cross-fire", those cells located in close proximity.