Cytogenetic analysis was used to test whether or not human bile induced chromosome abnormalities in lymphocytes grown in culture. Bile was obtained from gallbladders resected for various reasons such as cholecystitis, cholelithiasis, polypus and cancers of the biliary tract, stomach and pancreas. After adding human bile to a final concentration of 25 microliters/ml or 12.5 microliters/ml, the culture medium was incubated at 37 degrees C for 72 hr. Air-dried slides were stained with conventional Giemsa and the numerical and structural chromosome abnormalities were scored. Positive and negative controls in terms of chromosome abnormalities were established by using 0.03 micrograms/ml mitomycin C (MMC) and 0.9% normal saline, respectively. Cytogenetic analysis was successfully performed in 6 out of 10 bile samples (60.0%). Bile alone did not induce numerical or structural chromosome abnormalities. Structural abnormalities increased significantly in the 25 microliters/ml bile + 0.03 micrograms/ml MMC group, compared with the 0.03 micrograms/ml MMC group: 36.0% vs. 20.7% in the chromatid-type gaps and breaks, 27.8% vs. 22.7% in the chromosome-type gaps and breaks, and 8.3% vs. 3.2% in the exchange-type abnormalities. It is likely that the interaction between bile and MMC is synergistic rather than additive.