Recently, a novel procedure to isolate a highly pure and active Photosystem II preparation directly from thylakoid membranes, referred to as PS II-LHC II supercomplex, was reported [Eshaghi et al. (1999) FEBS Lett 446: 23-26]. In addition to the reaction center core proteins, the supercomplex contains all the extrinsic proteins of the oxygen evolving complex and a set of chlorophyll a/b binding proteins. In this paper, the functional properties of this isolated supercomplex are further characterized by using EPR spectroscopy, thermoluminescence, fluorescence relaxation kinetics and flash induced oxygen yield measurements. The PS II-LHC II supercomplex contains, in addition to Q(A) and Q(B), a small pool of plastoquinone (PQ). Although the isolated complex is no longer membrane bound, it has preserved functional characteristics of a well defined PS II preparation with the exception of some modification of Q(B) sites.