Two herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants with defects in the gene for the major capsid protein, ICP5, were examined for their effects on virion capsid assembly. Polyacrylamide gel electrophoresis revealed that both mutants were able to synthesize wild-type (WT) levels of ICP5 at the nonpermissive temperature. However, the 53 kD capsid protein disappeared concomitant with the appearance of a new, 51 kD species. These results, taken together with ultrastructural and immunological analyses indicate that the processing and assembly of capsid proteins, DNA packaging and thermal stability of HSV-1 virions are dependent upon functional ICP5.