We describe experiments showing that the 5'-flanking region of the human alpha-fetoprotein (AFP) gene contains transcription control elements with characteristics of enhancers. The enhancer activity was detected and characterized by the ability to direct the expression of a linked chloramphenicol acetyltransferase (CAT) gene in transfected AFP-producing hepatoma cells in culture. The enhancer activity is cell-specific in that it occurs in hepatoma cells producing AFP, but not in non-AFP-producing hepatoma or nonhepatic cells. The active elements can direct CAT expression in conjunction with the SV40 promoter in an orientation- and position-independent manner. The sequences important for enhancer activity reside in the 400-base pair region between 3.3 and 3.7 kilobase pairs upstream of the AFP gene. This and proximal upstream regions contain multiple enhancer "core"-like sequences and other stretches of potential biological significance.