The sensitivities of human breast cancer cells to hormones and chemotherapeutic agents were measured using a new in vitro assay. Tumor cells from individual patients were cultured on collagen-coated dishes in medium containing the patient's serum. The rationale for use of the patient's serum is that the components of this serum interact with the cells and therapeutic agents in vivo. Cells were incubated for the length of the assay in the presence or absence of estrogen (E2) with or without tamoxifen (TAM) or in the presence or absence of cortisol (F). At 1 day after cell seeding, cells were exposed to a chemotherapeutic agent, Adriamycin, melphalan, or 5-fluorouracil, for 24 h. After a 48-h recovery period, [3H]-thymidine ([3H]-TdR) was added to the cultures for 24 h. Depending on the concentration, E2 generally stimulated or inhibited incorporation of [3H]-TdR into the DNA of cells from estrogen-receptor (ER)-positive tumors. TAM eliminated the effects of E2. F generally stimulated or inhibited incorporation in cells with no correlation to ER status. Stimulation of [3H]-TdR incorporation by hormones increased cell sensitivity to Adriamycin. In contrast, hormone inhibition of [3H]-TdR incorporation decreased cell responsiveness to this drug. This rapid assay, which can measure the sensitivities of breast carcinoma cells to hormones and drugs and identify effective combinations of therapeutic agents, should lead to a rational selection of treatment for the individual patient.