Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from bulk DNA. Our results suggest that there are approximately 45,000 CpG islands per haploid genome in humans and 37,000 in the mouse. Sequence comparison confirms that about 20% of the human CpG islands are absent from the homologous mouse genes. Analysis of a selection of genes suggests that both human and mouse are losing CpG islands over evolutionary time due to de novo methylation in the germ line followed by CpG loss through mutation. This process appears to be more rapid in rodents. Combining the number of CpG islands with the proportion of island-associated genes, we estimate that the total number of genes per haploid genome is approximately 80,000 in both organisms.