Recent studies indicate that gene expression in higher eukaryotes is accompanied by a decrease in the frequency of 5-methyl cytosine residues around the activated site (Razin and Riggs, 1980). 5-aza-cytidine (5-aza-C) is an analogue that reduces cytidine methylation in DNA (Jones and Taylor, 1980) and has been reported to change the differentiation pattern of cultured mouse embryo cells (Taylor and Jones, 1979). We have tested its ability to activate the Epstein-Barr virus cycle in latently EBV-infected human lymphoid lines. After an incubation period of 6 to 8 h with the drug, early antigens (EA) were induced in a substantial fraction of the cells in all six lines tested that had a low rate of spontaneous viral antigen production. Optimal conditions for EA induction were defined. The efficiency of 5-aza-C was comparable to the inducing effect of iododeoxyuridine. EBV-DNA and EBNA positive virus-non-producer lines did not respond to 5-aza-C treatment. The findings are discussed in relation to the possibility that changes in EBV-gene expression may be related to the state of DNA methylation.