1-beta-D-Arabinofuranosylcytosine (ara-C) and aphidicolin are well known inhibitors of DNA synthesis, each one acting through a different mechanism. We have examined the effects of these compounds on DNA methylation in normal human embryonic lung fibroblasts (WI-38) as well as in their Simian Virus 40 (SVWI-38) and gamma radiation (CT-1) transformed counterparts. Analysis of the methylation status of the total genomic DNA in WI-38 cells revealed that hydroxyurea was the only drug which resulted in a significant increase in the 5-methylcytosine content under conditions where greater than 98% inhibition of DNA synthesis was achieved. Under the same conditions, all three drugs were capable of inducing hypermethylation in the SVWI-38 cells, whereas none of them showed any effect on the methylation status of the DNA in the CT-1 cells. In cells where a limited degree of replication was allowed to occur at drug concentrations resulting in 50% inhibition of DNA synthesis, a different pattern emerged. Under these conditions, DNA which was synthesized in the presence of either ara-C or aphidicolin was significantly hypermethylated in both the transformed cell lines, whereas hydroxyurea had no effect. In the normal WI-38 cells however, hydroxyurea was still the only drug which caused any significant hypermethylation. Different cells thus responded differently to these three agents, and the mere slowing down of DNA synthesis did not ipso facto lead to increased DNA methylation.