Previous investigations from our laboratory [Jabbari, K., Cacciò, S., Pais de Barros, J.P., Desgres, J., Bernardi G., 1997. Evolutionary changes in CpG and methylation levels in the genome of vertebrates. Gene 205, 109-118.] led to the discovery of two different methylation levels in the genomes of vertebrates, a higher one exhibited by fishes and amphibians and a lower one shown by mammals and birds. It was also noted that data from the literature indicated a higher CpG level in fishes and amphibians compared to mammals and birds. Such observations led to suggesting the existence of two equilibria and to speculate that the transitions between the two equilibria in DNA methylation and CpG levels were due to a higher deamination rate in warm-blooded vertebrates related to their higher body temperature. Here we used Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) analysis to study methylation levels in a number of fish genomes living at different temperatures. We found that polar fishes exhibit DNA methylation levels that are higher than those of tropical and temperate fishes, the latter being in turn higher than the methylation levels of warm-blooded vertebrates, as expected from previous work. A closer analysis of the data revealed that, among Antarctic fishes, the Channichthyidae (the icefishes, deprived of haemoglobin) had the highest methylation level, and that, among temperate and tropical fishes the latter showed the lowest methylation level. These results confirm the existence of an inverse relationship between DNA methylation and body temperature, when the latter is maintained over evolutionary times.