5-Azacytidine (5-AZC) reduces cytosine methylation in DNA and has been reported to activate quiescent virus genes. Treatment of explant cultures of latency herpes simplex virus type 2 (HSV-2)-infected guinea-pig dorsal root ganglia and spinal cords in vitro with 5-AZC significantly enhanced the rate of HSV recovery. Both the number of isolates from ganglia (P<0.001) and the rate of recovery (P<0.001) were significantly increased with the addition of 50 μM-5-AZC to explant cultures. Increased virus recovery appeared to be due to the induction of reactivation of latent virus, rather than an increase in replication, since 5-AZC inhibited HSV replication. These data support a role for methylation in HSV latency and reactivation.