By employing a procedure that combines ELISA and photoacoustic spectroscopy, we have examined the content of 5-methylcytosine (m⁵C) in DNA of individuals who differed from one another in the number of X chromosomes in their genomes. The results show that the human inactive X chromosome (Xi) contains very high amounts of this modified nucleotide. We estimate that in the 46, XX female there is more m⁵C in Xi (∼ 3.6 x 10⁷) than in all the remaining chromosomes put together (∼2.1 x 10⁷). Our results also suggest that nearly one-fifth of all cytosines in Xi are methylated and that, in addition to CpG methylation, there is extensive non-CpG methylation as well.