One basis for the evolution of organisms is the acquisition of new temporal and spatial domains of gene expression. Such novel expression domains could be generated either by cis sequence changes that alter the complement of trans-acting regulators binding to control elements or by changes in the expression patterns of one or more of the regulatory (trans) factors themselves. The gamma globin gene is a prime example of a gene that has undergone a distinct change in temporal expression at a defined time in evolution. Approximately 35-55 million years ago, the previously embryonic gamma gene acquired a fetal expression pattern. This change occurred in a simian primate ancestor after the separation of simian and prosimian primates but before the further separation of the major simian lineages; thus, the (prosimian) galago gamma gene retains the ancestral embryonic expression pattern, whereas the (simian) human gamma gene is fetal. This analysis of galago and human gamma genes in transgenic mice demonstrates that cis changes in sequences within a 4.0-kilobase region surrounding the gamma gene were responsible for the evolution of a novel fetal expression pattern in the gamma globin genes of simian primates.