We used molecular genetic methods to generate systematically altered forms of CCAAT/enhancer-binding protein (C/EBP). The aim of our experiments was to identify regions of C/EBP that contribute to its capacity to activate transcription from the promoter of the serum albumin gene in cultured hepatoma cells. Earlier experiments had shown that the DNA-binding domain must remain intact for C/EBP to activate albumin transcription. We now provide evidence of two additional elements of C/EBP that are required for its gene-activating role. One such element occurs within a 28-residue region located close to the amino terminus of the protein. The other maps to a broader, more internal region of the protein and appears to exhibit functional redundancy. These newly defined elements of C/EBP exhibit two characteristics of "activation" domains delineated in studies of other gene regulatory proteins. First, they play no obvious role in the capacity of C/EBP to bind to its DNA substrate. Second, they retain function after being appended onto the DNA-binding domain of a different protein. Neither of these putative activating elements is characterized by overt distinction in either charge or preponderance of any particular amino acid. The more amino-terminal element does, however, exhibit several features suggesting that it may assume an alpha-helical structure. These studies offer observations and reagents that will be valuable for future studies concerning the physiologic function of C/EBP.