The herpes simplex virus 1 genome consists of two components, L and S, each containing unique sequences flanked by inverted repeats. Each of the 6.5-kilobase pair inverted repeats of the S component, designated a'c' and ca, contains an approximately 700-base pair sequence (designated c1) located between the a sequence and the 3' terminus of the alpha 4 gene. Like the a sequence, c1 consists of direct repeats and unique sequences. Its function is not known. To probe for its function, we constructed a plasmid containing a viral thymidine kinase (TK) gene inserted into the c1 sequence. The construct was recombined into the genome of a TK- virus by cotransfection with intact viral DNA and selection for TK+ virus. As predicted from previous studies (Knipe et al., Proc. Natl. Acad. Sci. U.S.A. 75:3896-3900, 1978), the TK gene was found to be present in both copies of the c1 sequence in the R3104 virus. To delete the c1 sequence we constructed a plasmid containing 4 kilobase pairs of pBR322 flanked by an a sequence and by structural sequences of the alpha 4 gene. In this instance the cells were transfected with the construct and R3104 DNA; the progeny of the transfection was plated in the presence of 5-bromo-2'-deoxyuridine, and the selection was for TK- virus (R3158). The pBR322 DNA sequences replaced the c1 at both termini of the S component in R3158 DNA, but a sequence homologous to c1 was present in proximity to the 3' terminus of the alpha 4 gene. The results indicate that the c1 region has no significant role in the replication of the virus in cell culture. The advantage of inserting the pBR322 sequence is that it permits efficient cloning of large herpes simplex virus 1 DNA fragments by simple ligation of digests and transformation of appropriate Escherichia coli strains. The effortless selection of recombinants carrying inserts in both copies of the c1 restates the usefulness of this technique for selection of insertion deletion recombinants and underscores the rapid emergence of sequence identity at both ends of the reiterated regions of the S component as previously reported (Knipe et al., Proc. Natl. Acad. Sci. U.S.A. 75:3896-3900, 1978).