Effects of single and repeated doses of nilotinib on the pharmacokinetics of midazolam, a cytochrome P450 3A (CYP3A) substrate, were assessed in 2 separate studies. In the single-dose nilotinib study, 18 healthy subjects were randomized to 6 treatment sequences to receive single dose of nilotinib 600 mg, midazolam 4 mg, and coadministration of both in a crossover manner. In the repeated-dose nilotinib study, 19 chronic myeloid leukemia patients took a single dose of midazolam 2 mg on days 1 and 13, and nilotinib 400 mg twice daily from days 2–13. In the single-dose study, the geometric mean ratio of the area under the plasma concentration time curve extrapolated to infinity (AUCinf) of midazolam plus nilotinib vs. midazolam was 1.3 (90%CI, 1.2–1.5) and the maximum observed serum concentration (Cmax) was 1.2 (90%CI, 1.0–1.4). In the repeated-dose study, the values for AUCinf and Cmax were 2.6 (90%CI, 2.1–3.3) and 2.0 (90%CI, 1.7–2.4), respectively. These results indicate that single-dose and repeated-dose administration of nilotinib results in weak and moderate inhibition of CYP3A, respectively. Therefore, appropriate monitoring and dose adjustment may be needed for drugs that are mainly metabolized by CYP3A, and have narrow therapeutic index, when coadministered with nilotinib.