The formation of frog virus 3 virions takes place within morphologically distinct regions of the cytoplasm termed assembly sites. These sites are formed within infected BHK cells by 6-7 hr after infection, a time when viral DNA and both early and late proteins are present. To identify macromolecules involved in assembly site formation, a temperature-sensitive mutant ( ts9467 ) was used which is not only defective in the synthesis of late RNA and proteins (D.B. Willis, R. Goorha , and A. Granoff , 1979, Virology 98, 328-335), but, as reported here, also does not form assembly sites at nonpermissive temperatures. When ts9467 -infected cells were shifted from the nonpermissive to permissive temperature, assembly sites were observed within 1 hr even when late protein synthesis was inhibited by cycloheximide. Monoclonal antibodies specific for early and late viral proteins were used to show that assembly sites formed under these conditions contained at least one early protein, but lacked four representative late proteins. These results indicate that assembly site formation involves interaction between one or more early proteins and viral DNA, and that late proteins do not play a role in this process.