Cytoplasmic aggregates of ubiquitinated TAR DNA‐binding protein 43 (TDP‐43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP‐43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP‐43 on aggregate formation and fragmentation, using TDP‐43 expression constructs for WT or mutant TDP‐43 with a modified nuclear localizing signal (LQ‐NLS). Overexpression of the LQ‐NLS mutant alone induced no detectable cytoplasmic aggregates during a 72‐hr period. Polyubiquitination of both WT TDP‐43 and the LQ‐NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP‐43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ‐NLS mutant. Polyubiquitin‐charged WT and mutant TDP‐43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3‐methyladenine (3MA) blocked degradation of both TDP‐43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP‐43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP‐43 types. This is the first report delineating the distribution of polyubiquitinated TDP‐43 and the degradation pathway of TDP‐43 and clarifying the crucial role of autophagosomes in TDP‐43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP‐43 and the role of autophagosomes may help in understanding and treating ALS. © 2009 Wiley‐Liss, Inc.