How signaling molecules achieve signal diversity and specificity is a long-standing cell biology question. Here we report the development of a targeted delivery method that permits specific expression of homologous Ras-family small GTPases (i.e., Ras, Rap2, and Rap1) in different subcellular microdomains, including the endoplasmic reticulum, lipid rafts, bulk membrane, lysosomes, and Golgi complex, in rodent hippocampal CA1 neurons. The microdomain-targeted delivery, combined with multicolor fluorescence protein tagging and high-resolution dual-quintuple simultaneous patch-clamp recordings, allows systematic analysis of microdomain-specific signaling. The analysis shows that Ras signals long-term potentiation via endoplasmic reticulum PI3K and lipid raft ERK, whereas Rap2 and Rap1 signal depotentiation and long-term depression via bulk membrane JNK and lysosome p38MAPK, respectively. These results establish an effective subcellular microdomain-specific targeted delivery method and unveil subcellular microdomain-specific signaling as the mechanism for homologous Ras and Rap to achieve signal diversity and specificity to control multiple forms of synaptic plasticity.