Phenotypic analysis of the herpes simplex virus type 1 temperature-sensitive DNA-positive mutant, ts1233, revealed that the mutant had a structural defect at the nonpermissive temperature (NPT). Cells infected with ts1233 at the NPT contained large numbers of intermediate capsids, lacking dense cores but possessing some internal structure. No full capsids or enveloped virus particles were detected. In contrast to the defect in another packaging-deficient mutant ts1201, the block in the formation of dense-cored, DNA-containing capsids in ts1233-infected cells at the NPT could not be reversed by transferring the cells to the permissive temperature in the presence of a protein synthesis inhibitor. Furthermore, the capsids produced by ts1233 at the NPT had more compact internal structures than those of the gene UL26 mutant ts1201. Southern blot analysis of viral DNA in ts1233-infected cells confirmed that the mutant DNA was not encapsidated at the NPT and showed that the unpackaged DNA was not cleaved into genome-length molecules. The ts1233 mutation was mapped by marker rescue to the vicinity of genes UL32 and UL33. Sequence analysis of the DNA in this region from the mutant and two independently isolated revertants for growth revealed that ts1233 had a single base-pair change at the amino-terminal end of UL33, resulting in the substitution of an isoleucine with an asparagine. The nucleotide sequence of the revertants in this part of the genome was identical to that of wild-type virus.