Three distinct subpopulations of tumor cells derived from a single parent strain BALB/cfC3H mammary adenocarcinoma were tested in vivo for sensitivity to cyclophosphamide, methotrexate, and 5-fluorouracil. Treatment was begun either 2 days after s.c. tumor cell injection or at the time when the tumors became palpable. It was given on a weekly basis for 4 weeks. The mice were observed for growth of the primary implant and for development of spontaneous metastases. The three subpopulations differed markedly in their sensitivity to the drugs. The effects of the drugs ranged from induction of regression of the "primary" to enhancement of metastases. The effect on primary growth was independent of that on metastasis. The effect of the time of administration of the drugs also varied among the subpopulations. The sublines were also tested in vitro with methotrexate and 5-fluorouracil. Again there were marked differences in sensitivity to inhibition of cell division by the drugs. The relative sensitivities in vitro did not correlate with observations in vivo. The existence of subpopulations of tumor cells, differing in sensitivity to therapeutic agents, within a single neoplasm, presents a challenge to development of assays capable of predicting drug response and to the selection of combination therapies.