The induction of chromosomal aberrations by herpes simplex virus (HSV) and the interaction between viral deoxyribonucleic acid (DNA) and chromosomes have been studied (i) by infection of the BHK-21 line of hamster kidney cells at multiplicities ranging from 0.5 to 100 followed by 1-hr pulses of (3)H-thymidine preceding preparation at varying intervals of metaphases and autoradiography, and (ii) by use of (3)H-thymidine-labeled HSV for infection, chromosomal, and autoradiographic analyses at intervals thereafter. The results revealed that (i) chromosomal lesions develop prior to, and thus are independent of, viral DNA synthesis; (ii) HSV is capable of replicating in cells during the late G-2 period or in metaphase; (iii) most of the viral DNA remains unassociated with chromosomes and is not detectable at sites of chromosomal lesions; (iv) the capacity of the virus to cause chromosomal aberrations is four times less sensitive to inactivation by ultraviolet (UV) irradiation than its infectious property; and (v) after large doses of UV, invasion of the nuclei by the irradiated virus is reduced. These observations indicate that the chromosomal lesions induced by HSV result most likely from action of an early enzyme under control of the viral genome. This explanation is proposed also for the effects of adenovirus type 12 on chromosomes.