To investigate chromosomal site(s) of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in biochemically transformed [HeLa(BU25)/KOS 8–1] cells, these human cells which had been transformed by ultraviolet light-irradiated HSV-1 were fused with TK-negative mouse LM(TK−) cells, human-mouse somatic cell hybrid clones (LH81 clones 1–20) were isolated by HATG-ouabain selection and their chromosomes and isozymes were analyzed. Electrophoretic and serological analyses showed that all 20 clones expressed type-specific HSV-1 TK. Isozyme analyses on 29 gene-enzyme systems representing 22 human chromosomes revealed that all of the HSV-1 TK-positive clones expressed human aminoacylase-1 (ACY-1) and esterase D (ESD), which have been mapped to human chromosomes 3 and 13, respectively. Other human isozymes were detected in only one to four clones or in none of the clones. Chromosome analyses showed that: (1) the hybrid clones retained only a few human chromosomes; (2) a marker chromosome, designated M7, consisting of a chromosome 17 translocated to the short arm of chromosome 3, occurred in 36 out of the 41 metaphases examined of LH81-4 clones 1 to 4 and in 31 out of the 33 metaphases examined of LH81–12 clone 10; (3) a modified M7 chromosome, (M7/m), in which the distal 2/3 of the long arm of M7 was translocated to a small acrocentric mouse chromosome, was the only human chromosome found in metaphases of LH81–13 clone 17; and (4) an intact human chromosome 13 was not present in LH81–12 clone 10 or LH81–13 clone 17 cells. Counterselection with BrdUrd resulted in the isolation of subclones lacking HSV-1 TK, human ACY-1 and ESD, and the human marker M7 chromosomes. The experiments indicate that the HSV-1 TK gene is probably associated in HeLa (BU25)/KOS 8–1 cells with marker chromosome M7, but the possibility is not excluded that the segment of human chromosome 13 which codes for ESD is involved.