The psbH gene encodes a small protein which copurifies with photosystem 2. In the cyanobacterium Synechocystis sp. PCC 6803, psbH is located upstream of the cytochrome b6-f complex genes petC and petA. In striking contrast, in the genomes of plant chloroplasts, psbH is cotranscribed with petB and petD, encoding the other two major subunits of the cytochrome b6-f complex. We report that in Synechocystis sp. PCC 6803 monocistronic psbH and dicistronic petCA transcripts are probably initiated separately, each from DNA regions bearing some similarity to Escherichia coli sigma 70 promoters. Synechocystis sp. PCC 6803 psbH null mutants were generated by cartridge mutagenesis. Studies using a rapid screening procedure involving in situ complementation showed that the PsbH protein is not absolutely required for the assembly of a functionally active photosystem 2 complex since psbH insertion and deletion strains were able to grow photoautotrophically. The rate of photoautotrophic growth was, however, slower than the wild type, and studies of oxygen evolution, chlorophyll fluorescence, and thermoluminescence indicated that this reduction in growth rate is probably due mainly to an impairment in electron flow from QA to QB. We conclude, therefore, that the PsbH protein is not an absolute requirement for photosystem 2 activity but that it functions to optimize electron flow between the two secondary plastoquinone acceptors by interacting with the QB site on the D1 protein.