Analysis of sister chromatid exchanges (SCEs) is widely used as an assay for mutagenic carcinogens. Visualization of SCEs generally requires that the cells be cultured for 2 cycles of replication with the thymidine analog bromodeoxyuridine (BrdUrd). To see if incorporation of BrdUrd into chromosomal DNA influences the SCE response after treatment with chemical compounds, we have studied the effect of BrdUrd incorporation on SCEs induced by 5 different chemicals: bleomycin (BLM), which causes DNA single- and double-strand breakage; proflavine (PF), which intercalates into DNA; mitomycin C (MMC), a polyfunctional alkylating agent that cross-links DNA and also forms monoadducts; and 2 chemicals that do not appear to interact with DNA directly, aphidicolin (APC), an inhibitor of DNA polymerase alpha; and 3-aminobenzamide (3AMB), an inhibitor of poly-(ADP-ribose)-polymerase. Chemical treatment was for the first, second, or both cell cycles, and BrdUrd was present for the first or both cell cycles. All treatments with BLM, PF, or MMC increased the SCE frequency independently of the BrdUrd labeling protocol. With APC and 3AMB, on the other hand, only small increases in SCE frequency were observed when treatment was for the first cell cycle, but there were far greater increases when the chemical was present for the second or for both successive cell cycles. To further determine at which cycle SCEs were formed after continuous treatment of cells with BrdUrd and a test chemical, we also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) in tetraploid cells. Bleomycin, PF, and APC induced almost equal numbers of SCEs in both cell cycles, but MMC appeared to induce more SCEs in the second cycle than in the first. This is probably caused by long-lived lesions that induce SCEs. 3-Aminobenzamide, which does not form persisting lesions, also induced more single than twin SCEs, suggesting that this compound affects BrdUrd-substituted DNA differently than it does unsubstituted DNA. This type of interaction between a chemical and BrdUrdsubstituted DNA should be taken into consideration when SCE analysis is used as an assay system.