The chromosome damage induced by herpes simplex virus type 1 (HSV-1) in vitro was examined up to 6 h after infection using HT-1080 cells. Initial damage occurring within 3 h was specific, involving uncoiling of chromosome 1q12-21 and to a lesser extent the pericentric regions of chromosomes 9, 16 and satellited chromosomes. For the initial unwinding, synthesis of the immediate early class of HSV proteins needed to occur as was demonstrated using HSV-1 temperature-sensitive mutants tsK and tsB7 and two viral inhibitors, beta-propiolactone and psoralen plus long wavelength u.v. light. Later damage included chromatid breaks, acentric fragments and pulverization which did not take place until synthesis of delayed early proteins had begun.