We have examined the effect of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and cycloheximide on the CV-1 cell cytoskeleton. Within a few hours after producing an inhibition of cellular protein synthesis, all these agents specifically disrupted the organization of the vimentin filament system with no discernable effect on microtubules or microfilaments during the period of observation. Furthermore, just as the inhibition of protein synthesis by cycloheximide is reversible, so was the disruption of vimentin filaments by cycloheximide.