cAMP receptor protein (CRP) of Escherichia coli has been labeled covalently with two fluorescent reagents, N- (iodoacetylaminoethyl)-1-naphthylamine-5-sulfonate (1,5-IAENS) and dansyl chloride. Both AENS-CRP and dansyl-CRP are fully active in binding cAMP and promoting transcription of the gal operon. When cAMP is added to a solution of AENS-CRP, there is an increase in fluorescence intensity and a blue shift of the emission maximum of AENS-CRP, indicating a conformational transition of the protein. This conformational change is induced only by cAMP and its biologically active analogs (tubercidin 3′,5′-monophosphate and N6,O2′-dibutyryl-cAMP), but not by cyclic nucleotides (such as cGMP and 1,N6-etheno-cAMP) which are competitive inhibitors of cAMP. Thus, the observed conformational change of CRP induced by cAMP may be related to the cAMP dependent gene transcription. In addition, another specific conformational change of CRP occurs when CRP interacts with the lac operon in the presence of cAMP. This is demonstrated by a cAMP-sensitive blue shift and a quenching of the emission spectrum of AENS-CRP upon binding to λh80dlac DNA but not to λh80 DNA. Nanosecond fluorescence depolarization studies of dansyl-CRP reveal that addition of cAMP does not elicit association or dissociation of CRP.