The transcription factor CREB (cAMP responsive element binding protein) is activated by protein kinase A (PKA) phosphorylation of a single serine residue. To investigate possible mechanisms of CREB regulation by phosphorylation, we initiated a structural and biophysical characterization of the full-length, wild-type CREB protein, an altered CREB protein (CREB/SER) in which the three cysteine residues in the DNA-binding domain were replaced with serine residues and a truncated protein (ACT265) which encompasses the entire activation domain of CREB. Circular dichroism (CD) reveals that CREB and CREB/SER have identical secondary structures and contain approximately 20% alpha-helix, 9% beta-strand, 34% beta-turn, and 37% random coil structures. PKA phosphorylation does not alter the CD spectra, and therefore the secondary structure, of CREB or of CREB bound to DNA. Protease cleavage patterns indicate that PKA phosphorylation does not induce a global conformational change in CREB. Furthermore, PKA phosphorylation does not change the DNA binding affinity of CREB for either canonical or non-canonical CRE sequences as measured by a fluorescence anisotropy DNA binding assay. Since PKA phosphorylation of CREB results in its specific binding to the transcriptional co-activators CREB-binding protein and p300, we suggest that the PKA activation of CREB occurs by the production of specific, complementary interactions with these proteins, rather than through the previously proposed mechanisms of a phosphorylation-dependent conformational change or increased DNA binding affinity.