Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cells in situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form. The present work, using confocal microscopy and ultrastructural cytochemical techniques, demonstrates the presence of F-actin within interphase nuclei of intact, uncompromised, dorsal root ganglion neurons in vitro and in vivo. Labeling by FITC-phalloidin detected the presence of intranuclear F-actin adjacent to the nucleolar periphery in a small fraction of cells in vitro, an observation confirmed by three-dimensional reconstruction. Ultrastructural analyses of cells exposed to heavy meromyosin (HMM), showed the presence of typical "arrowhead" complexes. The observation that these complexes were associated with nucleoli confirms that the intranuclear ligand detected by FITC-phalloidin indeed represents F-actin. Postembedding labeling with HMM conjugated to 20-nm colloidal gold (HMM-Au20) resulted in labeling similar to that obtained with HMM. However, HMM-Au20 was found to label a much larger fraction of cells, both in vitro and in vivo, than did FITC-phalloidin or HMM. This finding indicates that labeling with HMM-Au20 more accurately reflects the extent of actin polymerization in nuclei. Results from double labeling with HMM-Au20 and an antibody to alpha-sarcomeric actin confirmed that only a small amount of nuclear actin is in the F-form. Together, these results represent a first ultrastructural demonstration of the presence of F-actin in nuclei of neurons. While the role of nuclear F-actin has yet to be defined, the results suggest that F-actin may represent a component of the molecular motor responsible for the dynamic positioning of specific chromatin domains into the tissue-specific, nonrandom patterns observed in many cell types.