BACKGROUND The issue on timing and number of bile sampling for exfoliative bile cytology is still unsettled. METHODS A total of 100 patients with cholangiocarcinoma undergoing resection after external biliary drainage were randomized into two groups: a 2-day group where bile was sampled five times per day for 2 days; and a 10-day group where bile was sampled once per day for 10 days (registered University Hospital Medical Information Network/ID 000005983). The outcome of 87 patients who underwent laparotomy was analyzed, 44 in the 2-day group and 43 in the 10-day group. RESULTS There were no significant differences in patient characteristics between the two groups. Positivity after one sampling session was significantly lower in the 2-day group than in the 10-day group (17.0 ± 3.7% vs. 20.7 ± 3.5%, P = 0.034). However, cumulative positivity curves were similar and overlapped each other between both groups. The final cumulative positivity by the 10th sampling session was 52.3% in the 2-day group and 51.2% in the 10-day group. We observed a small increase in cumulative positivity after the 5th or 6th session in both groups. CONCLUSIONS Bile cytology positivity is unlikely to be affected by sample time.