A study of the in vitro reconstitution of sugar beet cytochrome b(559) of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b(559) form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b(559). Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b(559) subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b(559)-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b(559)-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b(559). A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.