PURPOSE/OBJECTIVE(S) We report on our early experience of a phase I dose escalation study of single fraction stereotactic partial breast irradiation (S-PBI) for early-stage breast cancer after partial mastectomy using a robotic stereotactic radiation system. MATERIALS/METHODS Patient eligibility included DCIS or invasive epithelial histologies, AJCC clinical stage 0, I, or II with tumor size < 3 cm, and negative margins. Prior to simulation, 3-4 gold fiducials were placed around the lumpectomy cavity to be used for real-time tracking during treatments. Dose limiting toxicity (DLT) was defined as grade ≥ 3 toxicity by CTCAE (version 4) deemed definitely related to treatment for: skin, ribs/bone (fracture), pulmonary (radiation pneumonitis), or neurological (intercostal or brachial plexus nerves) or any grade 4 or 5 toxicity definitely attributed to therapy. Both patients and physicians completed baseline and subsequent cosmesis outcome questionnaires using a 4-point scale ranging from excellent, good, fair, or poor. Cohort 1 was 22.5 Gy, cohort 2 26.5 Gy, and cohort 3 30 Gy all delivered in a single fraction. Dose escalation was permitted provided that one or fewer of the first seven, or two or fewer of nine patients experienced a DLT within 90 days of treatment within each dose group. If more patients had DLT in a given dose cohort, the maximum tolerable dose (MTD) would have been exceeded. RESULTS From 4/2016 to 1/2021, 11, 9, and 10 patients were treated on cohorts 1, 2 and 3 respectively. Of these, data from 18 evaluable patients from cohorts 1 and 2 and 6 patients from cohort 3, with at least 12 months of follow up are reported. Median f/u for cohort 1 was 36 m (r 24-48m), cohort 2 was 18 m (r 18-24m), and cohort 3 12 m (r 1-18m). Average age was 66.8 (r 47-84). Histology included 11 DCIS, and 19 invasive carcinomas. Of 11 DCIS, 9 were ER+, and 2 were ER-. All 19 invasive tumors were ER+. 23/29 patients received endocrine therapy, and no patient received chemotherapy. No patients experienced grade 3 or higher treatment related toxicity in the acute period (≤ 90 days), and MTD was not reached. There were two delayed grade 3 toxicities, (dose cohort 1 and 2, one patient had breast pain, and another had mastitis at 12 months and 9 months). No patients experienced grade 4 or 5 toxic events. Five patients developed fat necrosis in all three cohorts. Physicians scored cosmesis excellent or good at last follow-up in (10/11) 90%, (7/8) 87.5%, and (6/6)100%, in cohort 1,2, and 3 respectively, while patients scored excellent or good in (11/11) 100%, 8/8 (100%), and 5/6 (83%) patients. There has been no report of disease recurrences. CONCLUSION Dose was escalated to 30 Gy in single fraction, and the majority of patients maintained good or excellent cosmetic outcome, without grade 3 or 4 toxicity. Continued analysis of all cohorts are in progress.