Targeted α-therapy (TAT) is a promising approach for radiopharmaceutical therapy with various molecular targets, but uncertainty exists as to whether the subcellular location of a target influences its cytotoxicity. We used a model system that provides a subcellular “zip code” for TAT to determine whether localizing α-emitters closer to the DNA increases their cytotoxic effect. Methods: Human pleural mesothelioma (I45) and ovarian adenocarcinoma (SKOV3) cell lines were engineered to express a fusion Escherichia coli dihydrofolate reductase-yellow fluorescent protein localized to the DNA, nucleus, cytoplasm, and plasma membrane. Subcellular TAT was achieved by targeting Escherichia coli dihydrofolate reductase with [²¹¹At]At-trimethoprim (TMP), an analog of the antibiotic TMP labeled with α-emitting ²¹¹At. This model system was characterized using confocal microscopy, flow cytometry, and radioligand binding assays. In vitro cytotoxicity of subcellular [²¹¹At]At-TMP therapy was measured, followed by Monte Carlo subcellular dosimetry. In vivo biodistribution and antitumor efficacy of [²¹¹At]At-TMP were measured. Results: [²¹¹At]At-TMP targeted at the DNA, followed by the nucleus, yielded the highest in vitro cytotoxicity per ²¹¹At decay. The cytotoxic advantage persisted even after normalizing to α-particle nuclear dose deposition using subcellular dosimetry, suggesting a significant cytotoxic contribution by the α-recoil, which is not considered in standard subcellular dosimetry calculations. Targeting of the plasma membrane caused at least comparable cytotoxicity to cytoplasmic targeting, suggesting a potential role of membrane damage-induced cytotoxicity. In vivo xenografts responded similarly to nuclear versus cytoplasmic [²¹¹At]At-TMP, supported by subcellular dosimetry that predicted the relevance of subcellular TAT for the treatment of individual tumor cells and small tumor cell clusters. Conclusion: An α-emitter’s proximity to the DNA yields higher cytotoxicity, which can guide future TAT drug development for improved treatment of microscopic metastases in addition to macroscopic disease, potentially leading to better clinical outcomes.