Purpose: To study the safety, tolerability, and pharmacokinetics of the selective tyrosine kinase inhibitor nilotinib as a single agent or in combination with imatinib in patients with advanced imatinib-resistant gastrointestinal stromal tumors. Experimental Design: A phase I intercohort dose-escalation trial was done in patients who received either ( a ) single agent nilotinib 400 mg twice daily or ( b ) escalating doses of nilotinib (200 mg once daily, 400 mg qd, or 400 mg bid) plus imatinib 400 mg bid (10- and 14-hour interval daily), or ( c ) nilotinib 400 mg bid plus imatinib 400 mg qd. Safety, pharmacokinetics, and tumor assessments were done. Results: Oral clearance (CL/F) of nilotinib was similar across the combination groups (mean CL/F, 19.1-25.6 L/h), and lower than in the single-agent cohort (mean CL/F, 35.6 L/h). A linear relationship between nilotinib daily dose and peak concentration was observed in the combination cohorts. Observed adverse events (AE) were mostly nonhematologic. Frequently reported AEs were rash (40%), fatigue (38%), abdominal pain (36%), and nausea (36%). Severe AEs (grade 3 or 4) included abdominal pain (13%) and rash (9%), the latter mainly with the combination. Thirty-eight patients had stable disease and two patients achieved partial response with a median progression-free survival of 134 days for the entire group. Conclusions: Nilotinib alone or in combination with imatinib was well tolerated overall and showed clinical activity in imatinib-resistant gastrointestinal stromal tumor patients. This phase I trial identified single-agent nilotinib 400 mg bid or combined with imatinib 400 mg qd as possible phase II doses for further evaluation. (Clin Cancer Res 2009;15(18):5910–6)