To analyze the site of integration of the herpes simplex virus type I (HSV-I) thymidine kinase (TK) gene in biochemically transformed human cells, TK-HeLa-(BU25) cells were transformed to the TK+ phenotype by a cloned, 2 kbp Pvull fragment of HSV-I DNA. The transformed cells [HeLa(BU25)/TF pAGO PP3] were fused with mouse LM(TK-) cells, and human-mouse somatic cell hybrid clones (LH PP3 clones 1, 2, 3, 5 and 6) were isolated in HATG-ouabain selective medium. The HeLa(BU25)/TF pAGO PP3 cells and the LH PP3 hybrid clones expressed HSV-I specific TK activity and a herpesvirus-associated nuclear antigen, and contained herpesvirus nucleotide sequences. Molecular hybridization experiments were carried out to map the HSV-I and flanking cellular nucleotide sequences in the biochemically transformed cells. These experiments demonstrated that the HSV-I nucleotide sequences were integrated at a single site, and that the same cellular nucleotide sequences flanked the viral DNA in transformed HeLa(BU25)/TF pAGO PP3 and LH PP3 clone 5 cells. TK- revertant subclones isolated by growing the LH PP3 clone 5 cells in BrdUrd (and diphtheria toxin) failed to form colonies in HATG medium, but retained HSV-I nucleotide sequences. Isozyme analyses on 21 gene-enzyme systems representing 21 human chromosomes revealed that all of the LH PP3 clonal lines expressed human hexosaminidase B, which has been assigned to chromosome 5, and all were sensitive to diphtheria toxin, which is also a marker for chromosome 5. Chromosome analyses showed that chromosome 5 was the nly human chromosome present in mitoses of LH PP3 clone 5 cells and that human chromosome 5 was present in most of the mitoses of LH PP3 clone 1, 2, 3, and 6 cells. The latter clones also contained 1 or 2 additional human chromosomes in some of the cells. As expected from the molecular hybridization analyses, TK- revertants of LH PP3 clone 5 cells retained portions of chromosome 5 and expressed human hexosaminidase B. The results indicate that HSV-I nucleotide sequences were stably integrated in the biochemically transformed cells, most likely in human chromosome 5.