An essential issue derived from the sequencing of the human and other genomes is the identification of gene regulatory elements. Using in vivo footprinting and expression analysis, here we show that mouse and human CpG island promoters at homologous genes have a completely different organization in terms of size and binding of transcription factors. Despite these species-specific differences, a unifying picture emerges from the precise confinement of protein--DNA interactions between the 5' boundary of the CpG islands and the transcription initiation site. This finding allows direct localization of promoters on genomic sequences and reveals a very high rate of variation and evolutionary divergence of mammalian regulatory regions. Our results also show that CpG island promoters associated with tissue-specific genes, such as the human alpha-globin, are bound by ubiquitous factors that allow a constitutive low level of expression in many cell types.