TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules.We have generated a mouse by N-nitrose-N9- ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7rsq1. In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7 rsq1 localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7rsq1 fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7wt/wt splenocytes. Thus, TLR7rsq1 is a bona fide phenocopy of the TLR7 null mouse. Because TLR7rsq1 binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7rsq1 mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.