Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash (Magyar et al. 2018). As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (~50%) and 167 ns (~30%), and a longer-lived component (~20%). This kinetics are attributed to re-reduction of P680 •+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 μs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (~60%) and 10 ns (~30%), attributed to recombination of the primary radical pair P680 •+ Pheo •-, and a small longer-lived component (~10%). These data confirm that only the first STSF is capable of generating stable charge separation-leading to the reduction of Q A ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.