Loxoribine (7‐allyl‐7,8‐dihydro‐8‐oxo‐guanosine) acts as synthetic adjuvant in anti‐tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll‐like receptor (TLR) 7/MyD88‐dependent signaling pathway. TLR7‐ and MyD88‐deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7‐deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R‐848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG‐DNA‐driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen‐associated molecular patterns in endosomal/lysosomal compartments.