The alpha-fetoprotein (AFP) gene is expressed abundantly in the fetal liver and transcriptionally repressed in the adult liver, but can be reactivated during liver regeneration and in liver tumors. Previous studies identified three enhancers, E1, E2, and E3, upstream of the mouse and rat Afp genes and a single enhancer upstream of the human gene. We have compared the sequences upstream of the rodent and primate AFP genes. Our analysis demonstrates that the previously identified human enhancer is the counterpart to mouse E2. This comparison also reveals that a functional primate counterpart to the rodent E1 is absent due to a deletion that removes the core region of this enhancer. Furthermore, our studies identify a novel human enhancer corresponding to rodent E3. Despite the overall similarity of E3 between human and mouse, we found differences in transcription factor binding sites between these species. A C/EBP binding site is conserved but two other motifs in rodent E3, one that binds orphan nuclear receptors and a second that binds FoxA proteins, are not conserved in humans. The human counterpart to the rodent FoxA site can bind COUP-TF factors. Despite the overall sequence similarity in E3 between mice and humans, the difference in factor binding sites in E3, as well as the absence of E1 in primates, indicates that different mechanisms regulate AFP transcription in these different species.