We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.