An abrupt change of nucleotide substitution patterns at the time of mammalian radiation The Human genome shows large regional variation in GC-content (from 30% to 60%) at scales exceeding hundreds of kilobases. This variation is due to differences in substitutional processes that affect evolution of both coding and noncoding sequences. The origin, timing, and implications of this large-scale GC variation remain controversial. To gain insight into this question, we use a novel computational approach to estimate substitutional patterns across the human genome at different ages going back approximately 200 Myr. We demonstrate that large-scale variation in GC-content in the human genome was generated through substitutional biases prior to the radiation of eutherian mammals. Approximately at the time of mammalian radiation, the patterns of substitutions changed abruptly and dramatically. The new mammalian pattern of substitution is expected to largely homogenize GC content across the human genome over time. Eutherian mammals are also predicted to share a newly evolved, 4- to 8-fold higher rate of methylation-induced cytosine transition in 5'-CG-3' pairs compared to the reptilian ancestor.