This Phase 1/2 study aimed to determine optimal doses of daunorubicin (DNR; mg/m2) and cytarabine (mg/m2) to be combined with fractionated doses of gemtuzumab ozogamicin (GO, Mylotarg®; 3 mg/m2 on day 1, 4, and 7) satisfying safety requirements. Three dose levels of DNR/AraC were investigated namely (45, 100), (60, 100), and (60, 200). Patients included were acute myeloid leukemia in first relapse, aged 50–70 years. Hematological recovery was 31 days for neutrophil and 32 days for platelet counts. A documented infectious episode > Grade 2 occurred in 11/20 patients (55%). None of the 20 patients had signs of veno-occlusive disease. Overall, eleven patients reached complete remission (CR), two CR with incomplete platelets recovery. The results showed that combination of fractionated GO doses with DNR at 60 mg/m2/d for 3 days and cytarabine at 200 mg/m2/d for 7 days is tolerable and could be further investigated in the front-line therapy.