The alpha-fetoprotein (AFP) gene, an important system for studying developmental and tissue-specific gene expression, is regulated mostly through the control of transcription. The promoter and cis-acting DNA elements which regulate the rat gene lie within a 7 kbp region upstream of the cap site. We have determined the sequence of this entire region. It contains several repetitive elements and a species-specific distribution of DNA methylation sites. We aligned our rat AFP sequence with fragmentary mouse and human AFP sequences to define blocks of highly conserved sequence, which we then analyzed for homology to known transcription regulatory sequences. Our analysis demonstrates that the regulatory region of the rat AFP gene is unusually complex.