We evaluated the effect of the demethylating agent hexamethylenebisacetamide on reactivation of latent herpes simplex virus type 2 (HSV-2) from guinea pig neural and extraneural tissues. Four explant cultures from the dorsal root ganglia of 42 latently infected guinea pigs and vaginal and cervical explant cultures from 33 animals were divided so that half received 5 mM of hexamethylenebisacetamide supplemented media and half media alone. HSV-2 was recovered earlier and from a greater percentage of treated cultures than controls. For example, seven days after explant, HSV-2 was recovered from 35 of 84 (42%) treated dorsal root ganglia cultures compared to seven of 84 control cultures (p less than 0.0001). Likewise, HSV-2 was recovered seven days after explant from 11 of 66 (17%) treated external genital skin cultures and 2 of 66 control cultures (p less than 0.009), Hexamethylenebisacetamide had no effect on productive HSV-2 infection in guinea pig dorsal root ganglia cultures. This study provides evidence for a role of demethylation in the reactivation of latent HSV from neural as well as peripheral tissues and suggests that latent virus exists at these sites in a similar state. Hexamethylenebisacetamide should be useful in studies of herpes virus latency because it decreases the time necessary to recover virus from latently infected tissues and enhances the recovery of virus.