Mammalian neurons and neuron-like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)-L, NF-M, NF-H, and peripherin. To determine whether these proteins are found within the same 10-nm filament, light and electron microscope immunocytochemistry using peripherin and NF-specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double-label immunofluorescence showed that peripherin and NF-L, -M, and -H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF-containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF-L or peripherin/NF-M/-H double-label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins are found within the same IF.