The biologically relevant interactions of a transcription factor are those that are important for function in the organism. Here, a transgenic rescue assay was used to determine which molecular functions of Drosophila CCAAT/enhancer binding protein (C/EBP), a basic region-leucine zipper transcription factor, are required for it to fulfill its essential role during development. Chimeric proteins that contain the Drosophila C/EBP (DmC/EBP) basic region, a heterologous zipper, and a heterologous activation domain could functionally substitute for DmC/EBP. Mammalian C/EBPs were also functional in Drosophila. In contrast, 9 of 25 single amino acid substitutions in the basic region disrupted biological function. Thus, the conserved basic region specifies DmC/EBP activity in the organism.