The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of ori(s) located within the repeated sequences flanking the short unique arm (U(s)), and one copy of ori(L) located within the long unique arm (U(L)). Precise localization and characterization of ori(L) have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to ori(s). In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.