Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and "transformed" cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK+ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK+ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK+ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.