Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of photosynthesis in cyanobacteria, algae, and plants. The unusual redox reactions in PSII often lead to damage, degradation, and reassembly of this molecular machine. To identify novel assembly factors, high sensitivity proteomic analysis of PSII purified from the cyanobacterium Synechocystis sp. PCC 6803 was performed. This analysis identified six PSII-associated proteins that are encoded by an operon containing nine genes, slr0144 to slr0152. This operon encodes proteins that are not essential components of the PSII holocomplex but accumulate to high levels in pre-complexes lacking any of the lumenal proteins PsbP, PsbQ, or PsbV. The operon contains genes with putative binding domains for chlorophylls and bilins, suggesting these proteins may function as a reservoir for cofactors needed during the PSII lifecycle. Genetic deletion of this operon shows that removal of these protein products does not alter photoautotrophic growth or PSII fluorescence properties. However, the deletion does result in decreased PSII-mediated oxygen evolution and an altered distribution of the S states of the catalytic manganese cluster. These data demonstrate that the proteins encoded by the genes in this operon are necessary for optimal function of PSII and function as accessory proteins during assembly of the PSII complex. Thus, we have named the products of the slr0144-slr0152 operon Pap (Photosystem II assembly proteins).