The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 microg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).