Toll-like receptors (TLRs) play an essential role in defense responses. Immune cells express multiple TLRs which are simultaneously activated by microbial pathogens. PRotein Associated with Tlr4 A (PRAT4A) is a chaperone-like endoplasmic reticulum (ER)-resident protein required for the proper subcellular distribution of multiple TLRs. PRAT4A(-/-) mice show impaired expression of TLR2/4 on the cell surface and the lack of ligand-induced TLR9 relocation from the ER to endolysosome. Consequently, TLR responses to whole bacteria as well as to TLR2, 4 and 9 ligands are impaired. We here compare the interaction of these TLRs with PRAT4A. Association of endogenous PRAT4A was easily detected only with TLR4. The TLR4 region responsible for strong interaction with PRAT4A is very close to the site necessary for interaction with MD-2. By using transient expression, we were able to detect PRAT4A interaction with TLR2 and TLR9. The PRAT4A single-nucleotide mutant replacing methionine 145 with lysine (M145K) associates with TLR9 but does not rescue ligand-dependent TLR9 trafficking. By contrast, the M145K mutant weakly, if at all, associates with TLR2 and TLR4. The M145K mutant appreciably rescues cell-surface TLR2 expression and its responses in PRAT4A(-/-) bone marrow-derived dendritic cells, whereas little if any rescue of cell-surface TLR4/MD-2 expression and its responses occurs. These results demonstrate that PRAT4A differentially interacts with each TLR and suggest that a single-nucleotide change in the PRAT4A gene influences not only the strength of TLR responses but can also alter the relative activity of each TLR.