Brain metastases are frequent in HER2-positive breast cancer. ONT-380 (tucatinib) is a potent selective inhibitor of HER2 with intracranial activity in preclinical models.This was a phase I study of tucatinib with trastuzumab, without chemotherapy, in patients with progressive, measurable HER2-positive brain metastases. The study tested two schedules of tucatinib: cohort A was twice-daily and cohort B was once-daily. The primary objective was determination of the maximum tolerated dose (MTD). Secondary endpoints included objective response (intracranial and extracranial) using modified RECIST and clinical benefit rate (CBR).Overall, 41 patients were enrolled (cohort A n=22, cohort B n=19). Patients had a median of 2 prior treatments for metastatic breast cancer and 83% had progressed after prior brain radiation. The MTD of tucatinib for cohort A was 300 mg twice-daily and for cohort B was 750 mg once-daily. The most common dose-limiting toxicities included thrombocytopenia and AST/ALT elevation. Grade 3/4 AST/ALT elevation occurred in 9/41 patients (22%). Intracranial responses were observed in two of 17 (12%) patients in Cohort A and one of 17 (6%) patient in Cohort B treated at the MTD. In cohort A, CBR at 16 weeks was 35% (n=6). In cohort B, CBR at 16 weeks was 53% (n=9). Of 15 patients overall who experienced clinical benefit, 12 (80%) had received prior neratinib and/or lapatinib. Median progression-free survival for cohorts A and B was 3.4 months and 4.1 months, respectively.The combination of tucatinib and trastuzumab is tolerable and demonstrated preliminary evidence of efficacy in patients with HER2-positive brain metastases.