The subcellular locations in infected Vero cells of Kunjin (KUN) virus core protein C and NS4B were analyzed by immunofluorescence (IF) and by immunoelectron microscopy using monospecific antibodies. Selection of appropriate fixation methods for IF showed that both proteins were associated at all times with perinuclear membranes spreading outward in a reticular pattern and they entered the nucleus late during the latent period. Subsequently NS4B was also dispersed through the nucleoplasm, while C appeared in the nucleolus and the nucleoplasm. These nuclear locations were confirmed by immunogold labeling of cryosections of infected cells at 24 hr postinfection. Labeling of NS4B in cryosections was especially enriched in the perinuclear membranes of the endoplasmic reticulum. When C and NS4B were each expressed separately in stably transformed cell lines, both cytoplasmic and nuclear localization was observed by IF and confirmed by immunoelectron microscopy. Thus the two proteins translocated to the nucleus independently of each other and of other viral proteins. Dual IF with antibodies to double-stranded RNA showed that cytoplasmic locations of C and NS4B were apparently associated in part with the sites of viral RNA synthesis which were resistant to solubilization by Triton X-100.