In situ hybridization, using a biotinylated clone of frog erythrocytic virus (FEV), was conducted to determine the location of viral sequences in bullfrog erythrocytes. FEV-specific hybridization signals were found to correspond to mature cytoplasmic viral particles and assembly sites. These data are consistent with electron microscopic observations of viral assembly in the erythrocyte cytoplasm. Although FEV has morphological and biochemical properties similar to frog virus 3, our data suggest that the site of DNA replication and assembly of FEV is more similar to that of the poxviruses.