Internal endonuclease restriction fragments of herpes simplex virus type 1 (HSV-1) representing 70% of the viral genome have been cloned in bacteriophage lambda (AR# Z01-CP-04914-02-LMV). These include even the large Eco Rl D,G,H and I fragments up to 17,000 b.p. in size. Each cloned fragment has been characterized by Southern transfer hybridization. More than 40 clones have been obtained of the heterogeneous defective DNA (dDNA) of HSV-l WES gt recombinant hybrids (AR# Z01-CP-05102-01-LMV). From endonuclease restriction digests, three major classes of dDNA have been identified. The heterogeneity in each of these classes appears to result from a novel joint formed between the rightmost end of HSV-l and a variable position within the S unique region of the HSV-l parent.