A topoisomerase activity is associated with herpes simplex virus type 1. The enzyme was recovered from purified virions which were disrupted with 6 M-guanidine-HCl followed by renaturation of extracted proteins. Based upon the following observations, the virion activity is classified as a type I topoisomerase: (i) the linking number of a unique DNA topoisomer is altered in steps of one; (ii) ATP and MgCl2 are not required for activity; (iii) the enzyme can be trapped in a covalent complex with DNA; (iv) the covalent linkage to DNA is through a 3' phosphoryl bond. A number of lines of evidence strongly indicate that the topoisomerase is external to the nucleocapsid. For example, the activity was released by treatment of intact virions with NP40, and subsequent washing steps extracted most residual activity. When guanidine extracts were prepared from nucleocapsids, topoisomerase activity was not detectable. Finally, DNA within the virion did not appear to contain covalently attached proteins with properties similar to topoisomerases. Thus, the enzyme appears to be a component of the envelope or tegument structure of the virion.