CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830–10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA]2−), iron complexed to ethylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine mesylate ([Fe-HDFO]+), and copper complexed to o-phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA] and [Fe-DTPA]2− of CRP-cAMP detected hypersensitivities in the DNA-binding F α-helix, the interdomain hinge, and the ends of the C α-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA]2− also detected reductions in cleavage in the D and E α-helices, which are involved in DNA recognition. Cleavage by [Fe-HDFO]+ of CRP-cAMP detected hypersensitivities in β-strand 8, the B α-helix, as well as in parts of the F and C α-helices. [Fe-HDFO]+ also detected protections from cleavage in β-strands 4 to 5 and their intervening loop, β-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E α-helices. Cleavage by [(OP)2Cu]+ of CRP-cAMP detected hypersensitivities in β-strands 9 and 11 as well as in the D and E α-helices. [(OP)2Cu]+ also detected protections in the C α-helix , the interdomain hinge, and β-strands 2–7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)2Cu]+, which detected minor protections in β-strands 3–7, the interdomain hinge, and the C α-helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction. Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone.