Background <p>Myeloid cells are heterogeneous cells that are critical for spontaneous choroidal neovascularization (CNV) in the <i>Vldlr</i>^{<i>−/−</i>} mouse model. However, the specific myeloid cell subtype necessary for CNV remains unknown.</p> Methods and results <p>To investigate the role of monocytes, we bred <i>Ccr2</i>^{<i>−/−</i>} and <i>Nr4a1</i>^{<i>−/−</i>} mice into the <i>Vldlr</i>^{<i>−/−</i>} background. We found that <i>Ccr2</i> and <i>Nr4a1</i> deficiency had no effect upon macrophage counts, CNV lesion number, or total CNV area. Next, we investigated the role of microglia by generating <i>Vldlr</i>^{<i>−/−</i>}<i>Tmem119</i>^{<i>CreER/+</i>}<i>Rosa26</i>^<i>DTR/+</i> mice. Diphtheria toxin (DT) treatment reduced macrophage counts at CNV lesions and CNV lesion number, but did not affect total CNV lesion area. To target microglia via a second strategy, we generated <i>Vldlr</i>^{<i>−/−</i>}<i>Cx3cr1</i>^<i>CreER</i><i>Csf1r</i>^<i>iDTR</i> mice and treated them with a single low dose of tamoxifen to target microglia without affecting choroidal macrophages. DT treatment in <i>Vldlr</i>^{<i>−/−</i>}<i>Cx3cr1</i>^<i>CreER</i><i>Csf1r</i>^<i>iDTR</i> mice decreased macrophage counts at CNV lesions and CNV lesion number but again had no effect upon total CNV lesion area. To target choroidal macrophages and microglia, we treated <i>Vldlr</i>^{<i>−/−</i>}<i>Cx3cr1</i>^<i>CreER</i><i>Csf1r</i>^<i>iDTR</i> mice with 9 tamoxifen treatments. DT-treated mice showed dramatic reductions in macrophage counts, CNV number, and total lesion area.</p> Conclusions <p>These data suggest that monocytes and monocyte-derived macrophages are dispensable, microglia are likely initiators for CNV development, and choroidal macrophages are potential key contributors to CNV growth and/or maintenance in the <i>Vldlr</i>^{<i>−/−</i>} model.</p>