Short-lived α-emitting isotopes individually conjugated to monoclonal antibodies have now reached human use, but little is still known about their toxicity. Use of antibody targetable ²²⁵Ac nanogenerators is a new approach in the field of α-immunotherapy offering the advantage of a 10-d half-life (t_1/2) and increased potency due to generation of 3 new atoms, yielding a total of 4 α-particles. However, the 3 α-emitting daughter elements generated have the potential for significant toxicity as these nuclides are no longer bound to the carrier IgG. Cynomolgus monkeys were used to evaluate the toxicity of prototype ²²⁵Ac nanogenerators. Monoclonal antibody HuM195 (anti-CD33) is the carrier for planned human clinical trials of ²²⁵Ac; there are no CD33 sites in cynomolgus monkeys. In one experiment, 2 monkeys received a single intravenous dose of ²²⁵Ac-HuM195 at 28 kBq/kg. This dose level is approximately the planned initial human dose. In another experiment, 2 animals received a dose escalation schedule of 3 increasing ²²⁵Ac-HuM195 doses with a cumulative activity of 377 kBq/kg. The whole-blood t_1/2 of ²²⁵Ac, ratios of ²²⁵Ac to its ultimate α-emitting daughter nuclide ²¹³Bi, generation of monkey anti-HuM195 antibodies (MAHA), hematologic indices, serum biochemistries, and clinical parameters were measured. Monkeys were euthanized and examined histopathologically when the dose escalation reached toxicity. The blood t_1/2 of ²²⁵Ac-HuM195 was 12 d, and 45% of generated ²¹³Bi daughters were cleared from the blood. MAHA production was not detected. Approximately 28 kBq/kg of ²²⁵Ac caused no toxicity at 6 mo, whereas a cumulative dose of ∼377 kBq/kg caused severe toxicity. In the cumulative dosing schedule, single doses of ∼37 kBq/kg resulted in no toxicity at 6 wk. After ∼130 kBq/kg were administered, no toxicity was observed for 13 wk. However, 28 wk after this second dose administration, mild anemia and increases of blood urea nitrogen and creatinine were detected. After administration of an additional 185 kBq/kg, toxicity became clinically apparent. Monkeys were euthanized 13 and 19 wk after the third dose administration (cumulative dose was 377 kBq/kg). Histopathologic evaluation revealed mainly renal tubular damage associated with interstitial fibrosis. ²²⁵Ac nanogenerators may result in renal toxicity and anemia at high doses. The longer blood t_1/2 and the lack of target cell antigens in cynomolgus monkeys may increase toxicity compared with human application. Therefore, a dose level of at least 28 kBq/kg may be a safe starting dose in humans. Hematologic and renal function will require close surveillance during clinical trials.