The deprotonation of naturally occurring zeaxanthin (Zea) radical cations (Zea(center dot+)) to form neutral radicals (#Zea(center dot)) and their involvement in the qE portion of nonphotochemical quenching (NPQ) was examined. The radical cations are weak acids, and readily deprotonate to a long-lived neutral radical (#Zea(center dot)) that could serve as long-lived quenching sites. When #Zea(center dot) is eventually neutralized and Zea is reformed in the presence of D2O, the Zea has an opportunity to undergo H/D exchange. This paper examines evidence for HID exchange specific to qE activity in Arabidopsis thaliana. We demonstrate that Zea(center dot+) formed chemically via oxidation of Zea by Fe(III) in the presence of D2O undergoes H/D exchange with a significant intensity increase of the M+1 (d(1)Zea) and M+2 (d(2)Zea) mass peaks in the mass spectrum. Then leaves from wild-type A. thaliana were infiltrated with either D2O or H2O and exposed to light. The carotenoids were extracted and analyzed via electrospray ionization liquid chromatography/mass spectrometry (LC/MS) to examine the mass peak distribution of Zea. Only leaves exposed to light intensity that triggers qE in A. thaliana (>300 mu E m(-2)s(-1)) showed HID exchange. This result suggests that #Zea(center dot) can form by the deprotonation of the weak acid Zea(center dot+) during qE, and its possible impact on qE must be considered.