Poly(A)-rich RNA has been isolated from Ehrlich ascites tumor (EAT) cells and translated in a rabbit reticulocyte cell-free system. The intermediate filament protein, vimentin, was found to be a major translation product. Fractionation of the poly(A)-rich RNA by sucrose gradient centrifugation showed that the vimentin mRNA had a sedimentation coefficient of about 18 S corresponding to a molecular size of about 2000 nucleotides. This means that it must possess significant non-coding regions. Vimentin synthesized in vitro was identical to native vimentin with regard to its precipitability with ammonium sulphate, extent of phosphorylation and susceptibility to digestion by the vimentin-specific, Ca2+-activated proteinase. Poly(A)-rich RNA was also isolated from a number of tissue-culture cells and rat liver, which contain varying amounts of vimentin in situ. It was found that the amount of vimentin synthesized by these RNA preparations in a rabbit reticulocyte cell-free system is proportional to the amount of vimentin detectable in situ, suggesting that the amount of cellular vimentin may be controlled at the level of transcription.