To obtain multicellular spheroids from MCF-7 human breast cancer cells we adhered to the following procedure: (a) limiting the adherence of cell to the substratum; (b) seeding more than the minimum number of cells; (c) guaranteeing the presence of estrogens in the culture medium. Charcoal-dextran (CD)-treated sera seemed to inhibit spheroid formation. A reduction in the concentration of CD-human sera (from 10% to 5%) added to phenol-red-free medium facilitated progress from cellular aggregates to multicellular spheroids. Once the spheroids became initiated, size increased at a rate that showed a good fit to a Gompertzian equation (A = 0.368 +/- 0.067 alpha = 0.065 +/- 0.013, r range = 0.890-0.989). Three different patterns of spheroid morphology and proliferative kinetic were defined: (a) spheroids with diameter less than 200 microns had a constant pattern of heterogeneity in the distribution of 3H-TdR-labelled cells and in the expression of estrogen receptors; (b) spheroids 250 to 700 microns in diameter showed a decrease in the proportion of 3H-TdR-labelled cells accompanying inward progression (50% in the outer shell, less than 10% in a cell layer located at a depth of 150 microns) while, at a depth of 170 microns, of signs of concurrent cellular degeneration and death were apparent; and (c) spheroids with a diameter of greater than 750 microns showed a crust of viable cells uniformly labelled with thymidine without impairment of the proportion of labelled cells when progressing inward from the spheroid crust. The larger the spheroid volume, the lower its growth fraction and the longer its volume doubling time. The hormone-dependence of MCF-7 cells in forming multicellular spheroids represents a unique experimental model for assessing estrogen action on cell organization and proliferation.