Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo(-)) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 μM) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 μM and 2 μM resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the 'variable' part of chlorophyll fluorescence (which is the recombination luminescence of the pair P680 (+) Pheo(-)). A 50% inhibition was induced by 5 μM K-15 and the effect was maximal in the range 20 to 200 μM. Photooxidation of P680 in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 μM). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo(-) and P680 (+) (or Z(+)) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for QA missing in the preparation.At a concentration of 0.5-50 μM K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl2. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo(-) to cytochrome b559.