We previously selected two melanin-targeting radioligands [I-125]ICF01035 and [I-125]ICF01040 for melanoma-targeted I-125 radionuclide therapy according to their pharmacological profile in mice bearing B16F0 tumors. Here we demonstrate in vitro that these compounds present different radiotoxicities in relation to melanin and acidic vesicle contents in B16F0, B16F0 PTU and A375 cell lines. ICF01035 is effectively observed in nuclei of achromic (A375) melanoma or in melanosomes of melanized melanoma (B16F0), while ICF01040 stays in cytoplasmic vesicles in both cells. [I-125]ICF01035 induced a similar survival fraction (A(50)) in all cell lines and led to a significant decrease in S-phase cells in amelanotic cell lines. [I-125]ICF01040 induced a higher A(50) in B16 cell lines compared to [I-125]ICF01035 ones. [I-125]ICF01040 induced a G2/M blockade in both A375 and B16F0 PTU, associated with its presence in cytoplasmic acidic vesicles. These results suggest that the radiotoxicity of [I-125]ICF01035 and [I-125]ICF01040 are not exclusively reliant on DNA alterations compatible with gamma rays but likely result from local dose deposition (Auger electrons) leading to toxic compound leaks from acidic vesicles. In vivo, [I-125]ICF01035 significantly reduced the number of B16F0 lung colonies, enabling a significant increase in survival of the treated mice. Targeting melanosomes or acidic vesicles is thus an option for future melanoma therapy.