The cAMP response element binding protein CREB activates the transcription of genes in response to phosphorylation by cAMP-dependent protein kinase A (PKA) and other protein kinases. Phosphorylated CREB activates transcription by recruiting transcriptional co-activators such as the CREB binding protein. Here, we describe experiments that analyze the effects of phosphorylation on the DNA binding affinity of CREB and the structural characteristics of the CREB/DNA complex in solution. Analysis of deletion mutants of CREB indicate that amino acid sequences within the transactivation domain promote high-affinity binding of CREB to fluorescently labeled oligonucleotides containing cAMP response elements. In vitro experiments indicate that phosphorylation is processive between PKA as the initial kinase and glycogen synthase kinase-3 (GSK-3) but not casein kinase II as the secondary kinase. Fluorescent electrophoretic mobility shift assays show that phosphorylation by PKA results in a 3-5-fold increase in the binding affinity of CREB to both the symmetrical somatostatin CRE (SMS-CRE) and the asymmetric somatostatin upstream element (SMS-UE). Processive phosphorylation of CREB by GSK-3 attenuates the enhanced DNA binding in response to PKA thus acts as an inhibitor of PKA-induced binding. Ferguson plot analyses demonstrate that phosphorylation of CREB by PKA and GSK-3 result in an increase in the spherical size and the net positive surface charge of the CREB/DNA complex. Moreover, these analyses uncovered the unexpected finding that CREB associates as a tetramer both in the presence and absence of DNA. These findings suggest a model by which phosphorylation of CREB alters the secondary structure and charge characteristics of the CREB/DNA complex resulting in an alteration in binding affinity.