Abstract Toll-like receptor 3 (TLR3) senses dsRNA in the acidic environment of endosomes and plays a pivotal role during infection of the brain with viruses, including herpes simplex virus type 1 (HSV-1). Studies on various cell lines corroborate TLR3 presence in LAMP1-labeled endosomes following the receptor stimulation. However, co-localization times, as well as the subjection of co-localization on the TLR3 stimulation, remain divergent depending on the cell type. In this work, we examined TLR3 residence in the LAMP1-marked endosomes in nine different time intervals following poly(I:C) stimulation of C8-D1A murine astrocytes. Interestingly, TLR3 did not localize in the lysosomal compartment in resting cells, however, the presence of the receptor was observed in LAMP1-labeled endosomes from the first minute after the addition of poly(I:C). Furthermore, 24 h following stimulation, we observed numerous lysosomes co-localizing with TLR3, which were present in all astrocytes. Our results indicate LAMP1-labeled endosomes as important organelles which may serve as the TLR3 ligand recognition sites, however, further studies on the migration of dsRNA in astrocytes are essential.