AbstractShort polypeptides from intermediate filament (IF) proteins containing one of the two IF‐consensus motifs interfere severely with filament assembly in vitro. We now have systematically investigated a series of larger fragments of the muscle‐specific IF protein desmin representing entire functional domains such as coil1 or coil 2. “Half molecules” comprising the amino‐terminal portion of desmin, such as DesΔC240 and the “tagged” derivative Des(ESA)ΔC244, assembled into large, roundish aggregates already at low ionic strength, DesΔC250 formed extended, relatively uniform filaments, whereas DesΔC265 and DesΔC300 were soluble under these conditions. Surprisingly, all mutant desmin fragments assembled very rapidly into long thick filaments or spacious aggregates when the ionic strength was raised to standard assembly conditions. In contrast, when these desmin mutants were assembled in the presence of wild‐type (WT) desmin, their assembly properties were completely changed: The elongation of the two shorter desmin fragments was completely inhibited by WT desmin, whereas DesΔC250, DesΔC265 and DesΔC300 coassembled with desmin into filaments, but these mixed filaments were distinctly disturbed and exhibited a very different phenotype for each mutant. After transfection into fibroblasts and cardiomyocytes, the truncated mutant Des (ESA)ΔC244 localized largely to the cytoplasm, as revealed by a tag‐specific monoclonal antibody, and also partially colocalized there with the collapsed endogenous vimentin and desmin systems indicating its interference with IF‐organizing processes. In contrast, in cells without an authentic cytoplasmic IF system such as line SW13, Des(ESA)ΔC242 entered the nucleus and was deposited in small dot‐like structures in chromatin‐free spaces without any noticeable effect on nuclear morphology. Cell Motil. Cytoskeleton 66: 986–999, 2009. © 2009 Wiley‐Liss, Inc.