The effects of various concentrations of sodium butyrate were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40-transformed line (SVWI-38) and a cell line transformed by gamma-irradiation (CT-1). The rate of thymidine incorporation into DNA was inhibited by 60-80% in the WI-38 cells, even at butyrate concentrations as low as 5 mM. The two transformed cell lines showed no inhibition of DNA synthesis, even at concentrations of 75 mM butyrate. Analysis of RNA and protein synthesis revealed that the former was inhibited by +/- 20% at 5-10 mM butyrate in the normal WI-38 cell line, while protein synthesis was not inhibited at these concentrations. The inhibition of RNA synthesis was not dose dependent up to butyrate concentrations of 20 mM, and protein synthesis was inhibited less than 15% at this concentration. None of these inhibitory effects was observed in the case of the SVWI-38 or CT-1 cell lines. Analysis of the 5-methylcytosine content of DNA that was labeled either prior to or during treatment with butyrate revealed an increased content of methylcytosine when compared with control cells. Both preexisting and newly synthesized DNAs were thus subject to hypermethylation. Although all three cell lines showed a dose-dependent hypermethylation of DNA, the extent of this methylation differed in the normal and transformed lines, as preexisting DNA was more methylated in WI-38 cells compared with SVWI-38 and CT-1 cells, while methylation of newly synthesized DNA occurred to a greater extent in the SVWI-38 cells. These studies show that sodium butyrate affects major macromolecular synthetic processes as well as DNA methylation quite differently in normal and transformed cells.