Abstract Radioligand therapy (RLT) with prostate-specific membrane antigen (PSMA)-targeting ligands is effective in ~50% of patients with advanced prostate cancer (PC). Causes for RLT failure are not well understood. The prevalent PSMA heterogeneity in PC might contribute to RLT failure. Here we investigate the relationship between RLT efficacy and PSMA levels per cell, and PSMA heterogeneity. PC cells expressing different levels of PSMA (RM1-PSMA low, medium, high, or RM1-YFP that do not express PSMA), or a mix of PSMA− and PSMA+ cells (RM1-YFP/ RM1-PSMA-high; PC3/ PC3-PIP) at various ratios were subcutaneously injected into NSG mice. Mice were treated with 177Lu- or 225Ac-PSMA617 (RLT) and tumor growth was monitored. In a subset of mice, radioligand uptake (γ-counting), DNA damage and PSMA expression (anti-53BP1 and -PSMA immunohistochemistry, respectively) were quantified in tumors resected 2 days after RLT. Increasing PSMA levels and fractions of PSMA+ cells improved RLT efficacy in both, the RM1 and PC3 model. PSMA expression correlated with radioligand uptake into the tumor and the degree of DNA damage and. Treatment with 225Ac-PSMA617 (vs. 177Lu-PSMA617) improved RLT outcomes and tended to enhance the differences in therapeutic efficacy between experimental groups. Taken together, we demonstrate in mouse models of PC that optimal anti-tumor efficacy of RLT hinges on homogenously high target expression. Although PSMA-RLT is effective even in tumors with low PSMA levels or with a small number of PSMA+ cells, low or heterogeneous PSMA expression might result in undertreatment and selection of treatment resistant clones. Systematic assessment of intra- and inter-lesion PSMA heterogeneity is currently not feasible clinically; however, this issue might be addressed by individual patient dosimetry to optimize safely delivered maximal tumor doses. Clinical studies designed to determine intra- and inter-lesion PSMA heterogeneity and to optimize PSMA-RLT for each patient are highly warranted. Citation Format: Katharina Lueckerath, Kyle Current, Catherine Meyer, Clara Magyar, Christine E. Mona, Joel Almajano, Roger Slavik, Andreea D. Stuparu, Chloe Cheng, David Dawson, Caius Radu, Johannes Czernin. Heterogeneous tumor PSMA expression represents a resistance mechanism to PSMA-targeted radioligand therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5345.