The topography of the heme prosthetic group of cytochrome b-559 of the photosystem II reaction center was determined from measurement of the orientation of its alpha- and beta-polypeptides in thylakoid membranes of spinach chloroplasts and in osmotically disrupted cells of the cyanobacterium Synechocystis sp. PCC 6803. The accessibility to trypsin proteolysis of an epitope located near the solvent-exposed N-terminus of the beta-subunit was compared to that of the alpha-subunit, whose N- and C-termini had previously been localized from the trypsinolysis pattern to the stromal and lumenal sides of spinach thylakoid membranes, respectively [Tae et al. (1988) Biochemistry 27, 9075-9080; Vallon et al. (1989) Biochim. Biophys. Acta 975, 132-141]. The N-terminal epitope of the cyanobacterial beta-subunit was modified by introducing a tridecapeptide epitope, previously found to be immunoreactive, from the C-terminal region of the spinach chloroplast alpha-subunit. This epitope had no homology with the cyanobacterial alpha-subunit. The cells with the hybrid beta-subunit retained full photosynthetic activity. The intactness of membranes from osmotically shocked cyanobacteria was tested by trypsin inaccessibility to (a) the alpha-subunit C-terminus and (b) the manganese-stabilizing protein (MSP) of the oxygen-evolving complex that is on the lumenal side of the membrane. The loss after trypsinolysis of most of the beta-subunit immunoreactivity, under conditions where (i) the alpha-subunit was cleaved near the N-terminus in both spinach thylakoids and osmotically shocked cyanobacterial membranes and (ii) the MSP protein in cyanobacteria was not disrupted, implied that the orientation of the beta-subunit was parallel to that of the alpha-subunit in both kinds of membranes.(ABSTRACT TRUNCATED AT 250 WORDS)