Variable chlorophyll a (Chl a) fluorescence is composed of a photochemical and a thermal phases of similar amplitudes. The photochemical phase can be induced by a saturating single turnover flash (STF) and reflects the reduction of the Photosystem II (PS II) Q(A) primary electron acceptor. The thermal phase requires multiple turnover flash (MTF) and is somehow related to the reduction of the plastoquinone (PQ) molecules. This article aimed to determine the relative contributions of the Q(B)-bound and the free oxidized PQ molecules to the thermal phase of Chl a fluorescence. We thus measured the interactive effects of exogenous PQ (PQex), of an inhibitor (DCMU) acting at the Q(B) site of PS II and of an artificial quencher, 2-methyl-1,4-naphtoquinone, on Chl a fluorescence levels induced by STF (F(F)) and MTF (F(M)) in spinach thylakoids. We observed that: (1) the incorporation of PQex in thylakoids stimulated photosynthetic electron transport but barely affected F(F) and F(M) in the absence of DCMU; (2) DCMU significantly increased the amplitude of F(F) but slightly quenched F(M); (3) 2-methyl-1,4-naphtoquinone quenched F(M) to a larger-extent than F(F); (4) DCMU increased the quenching effects of PQex on F(F) and F(M) and also, of methyl-1,4-naphtoquinone on F(F). These results indicate that: (1) the Q(B)-bound and the free PQ molecules contribute to about 56% and 25%, respectively, to the thermal phase Chl a fluorescence in dark-adapted thylakoids; and (2) the thermal phase of Chl a fluorescence is more susceptible than the photochemical phase to the non-photochemical quenching effect of oxidized quinones.