TDP-43 aggregates (skeins and round inclusions [RIs]) are frequent histopathological features of amyotrophic lateral sclerosis (ALS). We have shown that diffuse punctate cytoplasmic staining (DPCS) is the earliest pathologic manifestation of TDP-43 in ALS, corresponding to nonfibrillar TDP-43 located in the rough endoplasmic reticulum. Previous in vitro studies have suggested that TDP-43 inclusions may be derived from stress granules (SGs). Therefore, we investigated the involvement of SGs in the formation of TDP-43 inclusions. Formalin-fixed spinal cords of six ALS patients with a disease duration of less than 1 year (short duration), eight patients with a disease duration of 2-5 years (standard duration), and five normal controls were subjected to histopathological examination using antibodies against an SG marker, HuR. In normal controls, the cytoplasm of anterior horn cells was diffusely HuR-positive. In short-duration and standard-duration ALS, the number of HuR-positive anterior horn cells was significantly decreased relative to the controls. DPCS and RIs were more frequent in short-duration ALS than in standard-duration ALS. The majority of DPCS areas and a small proportion of RIs, but not skeins, were positive for HuR. Immunoelectron microscopy showed that ribosome-like granular structures in DPCS areas and RIs were labeled with anti-HuR, whereas skeins were not. These findings suggest that colocalization of TDP-43 and SGs occurs at the early stage of TDP-43 aggregation.