We have examined structural differences between the proto-oncogene c-Myb and the cyclic AMP-responsive factor CREB that underlie their constitutive or signal-dependent activation properties. Both proteins stimulate gene expression via activating regions that articulate with a shallow hydrophobic groove in the KIX domain of the coactivator CREB-binding protein (CBP). Three hydrophobic residues in c-Myb that are conserved in CREB function importantly in cellular gene activation and in complex formation with KIX. These hydrophobic residues are assembled on one face of an amphipathic helix in both proteins, and mutations that disrupt c-Myb or CREB helicity in this region block interaction of either factor with KIX. Binding of the helical c-Myb domain to KIX is accompanied by a substantial increase in entropy that compensates for the comparatively low enthalpy of complex formation. By contrast, binding of CREB to KIX entails a large entropy cost due to a random coil-to-helix transition in CREB that accompanies complex formation. These results indicate that the constitutive and inducible activation properties of c-Myb and CREB reflect secondary structural characteristics of their corresponding activating regions that influence the thermodynamics of formation of a complex with CBP.