Recognition of nucleic acids by TLR9 requires its trafficking from the endoplasmic reticulum to endolysosomal compartments and its subsequent proteolytic processing. Both processes depend on interactions of TLR9 with the polytopic endoplasmic reticulum-resident protein UNC93B1. To examine the intracellular behavior of TLR9 in primary APCs, we generated transgenic mice expressing a TLR9-GFP fusion. The TLR9-GFP transgene is functional and is proteolytically processed in resting bone marrow-derived macrophages (BMDMs), dendritic cells, and B cells. Inhibition of cleavage impairs TLR9-dependent responses in all primary APCs analyzed. The kinetics of TLR9-GFP processing in BMDMs and B cells differs: in B cells, proteolysis occurs at a faster rate, consistent with an almost exclusive localization to endolysosomes at the resting state. In contrast to the joint requirement for cathepsins L and S for TLR9 cleavage in macrophages, TLR9-GFP cleavage depends on cathepsin L activity in B cells. As expected, in BMDMs and B cells from UNC93B1 (3d) mutant mice, cleavage of TLR9-GFP is essentially blocked, and the expression level of UNC93B1 appears tightly correlated with TLR9-GFP cleavage. We conclude that proteolysis is a universal requirement for TLR9 activation in the primary cell types tested, however the cathepsin requirement, rate of cleavage, and intracellular behavior of TLR9 varies. The observed differences in trafficking indicate the possibility of distinct modes of endosomal content sampling to facilitate initiation of TLR-driven responses in APCs. The Journal of Immunology, 2013, 190: 695-702.