The ts mutant F1 of vesicular stomatitis virus, New Jersey serotype, directs the synthesis of undermethylated 5'-terminal cap structures in vitro. In order to determine the relationship between the ts and hypomethylation phenotypes, a spontaneous revertant rev(ts)F1 of the ts phenotype was analyzed. The revertant retained the hypomethylation phenotype. The four cap structures (GpppA, 7mGpppA, GpppAm, and 7mGpppAm) synthesized in mutant and revertant-directed reactions in the presence of low as well as high concentrations of AdoMet were resolved by HPLC. Quantitation of the data and analysis of cap substrate to product ratios revealed that despite apparent similarities between the two hypomethylation phenotypes, the functional lesions in F1 and rev(ts)F1 were different. F1 displayed an AdoMet concentration-dependent alteration in the GpppA----GpppAm reaction and an AdoMet concentration-independent alteration in the GpppA----7mGpppA reaction. In contrast, rev(ts)F1 displayed AdoMet concentration-dependent alterations in the reactions GpppA----7mGpppA and GpppAm----7mGpppAm.