The human prostatic carcinoma cell line DU 145 was grown as multicellular spheroids in vitro. The volume doubling time during the early exponential growth phase was about 5 days. The saturation volume, in the plateau phase of the growth curve, was in the order of 1.4 mm3. The spheroids developed a central degenerative region surrounded by a 0.1-0.3 mm layer of viable cells. The DU 145 spheroid system is planned to be used as a model in studies on chemotherapy and targeted radiotherapy of micrometastases of prostatic cancer. Some effects of the drug estramustine, EM, a conjugate of estradiol and nornitrogen mustard, were analysed in this introductory study. Tritium-labelled estramustine, 3H-EM, bound both in the viable cell layers and in the degenerative region of the spheroid already after 1 hour of incubation which indicated good penetration. The viable cells bound only low levels of 3H-EM while the degenerative region bound 3H-EM to a higher extent. The amount of bound 3H-EM increased after incubation for 24 hours. The binding was nonspecific since it could not be inhibited by pretreatment with an excess of non-radioactive EM. Furthermore, 3H-EM bound to a similar extent in glioma and colon carcinoma spheroids used for comparison. Incubation of DU 145 spheroids for 24 hours with EM (20 mg/ml) induced a growth delay of 6-7 days and a transient increase in the volume of the extracellular spaces for a few days following the treatment. The results showed that the binding of EM to prostate DU 145 cells growing as spheroids was not specific and that the toxic action was limited. An interesting result was that EM works as an extracellular space expander. This might be exploited in combination treatments with other agents.