Two cDNA clones fully encoding the rat neurofilament proteins NF-L and NF-M were subcloned into eukaryotic expression vectors behind the strong constitutive viral promoters from SV40 and Rous sarcoma viruses. Transient transfection of L tk- and Cos cell lines with these expression constructs resulted in cells expressing the neurofilament proteins in an intermediate filament-type pattern. Additionally, a putative juxtanuclear organizing center or region was observed in the transfected cells, most noticeable shortly after the transfection procedure. Stable transfections were performed on mouse L tk- and Swiss 3T6 cells using NF-L and NF-M constructs bearing an SV40 early promoter driven neomycin selectable marker. Although G418-resistant clones were recovered with both the NF-L and the NF-M constructs, only clones expressing immunofluorescently stainable amounts of NF-M were detected and established. Immunoelectron microscopic analysis revealed NF-M and vimentin proteins to be colocalized on the same intermediate filaments.