The rate of repopulation of surviving cells increases during fractionated radiotherapy and limits the ability to control tumors. Repopulation is likely to be more important during the longer intervals between courses of chemotherapy and is a potential cause of clinical resistance to chemotherapy. To evaluate the rate of repopulation of surviving cells in MXT and EMT-6 mouse breast tumors between successive cycles of chemotherapy, tumor-bearing mice were treated with up to three weekly cycles of cyclophosphamide or 5-fluorouracil (5FU). Animals were killed at different intervals during and after chemotherapy, and uptake of 5'bromodeoxyuridine and immunohistochemical staining were selected to study repopulation. When tumors regrew after a single treatment, the proliferative rate of tumor cells returned to control values. During successive courses of chemotherapy, the proliferative rate of surviving cells increased in both tumors, e.g., in 5FU-treated MXT tumors, the percentages of 5'bromodeoxyuridine-labeled proliferating cells at 7 days after the first, second, and third treatment were 25 +/- 12%, 29 +/- 9%, and 27 +/- 11%; it was 32 +/- 6% at 14 days after the third treatment. The corresponding value in control tumors was 19 +/- 2% (P < 0.05 for each comparison). Accelerating repopulation after sequential treatments with cyclophosphamide or 5FU can lead to effective drug resistance in the absence of changes in the sensitivity of constituent cells.