The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FAD(ox), ca. 38.5%), in the semiquinone form (FADH, ca. 5%), and in the fully reduced neutral form (FAD(red)H(2)) or fully reduced anionic form (FAD(red)H(-), ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. Forster-type energy transfer from MTHF to FAD(ox) is observed. Photoexcitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FAD(ox) to FAD(red)H(2) (or FAD(red)H(-), FAD(ox), photo-cycle of moderate efficiency), reversible reduction of FADH(.) to FAD(red)H(2) (or FAD(red)H(-), FADH(.) photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H(2) (or FAD(red)H(-)) to permanent FAD(red)H(2) (or FAD(red)H(-)) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF', MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF"). (c) 2006 Elsevier B.V. All rights reserved.