PsaK and PsaM are small, integral membrane proteins, which are associated with the Photosystem I complexes of cyanobacteria. The complete genome sequence of Synechocystis sp. PCC 6803 has revealed the presence of two unlinked psaK genes: psaK1 (ssr0390) and psaK2 (sll0629). To investigate structural and functional roles of the PsaK1, PsaK2 and PsaM polypeptides in Synechocystis sp. PCC 6803, we generated targeted mutants that lack the functional psaK1, psaK2 or psaM genes. Inactivation of psaK1, psaK2 or psaM did not affect photoautotrophic growth, photosynthetic activity and accumulation of other subunits of the Photosystem I complex. The psaK1 (-), psaK2 (-) and psaK1 (-) psaK2 (-) mutants showed normal levels of Photosystem I trimers, whereas the lack of PsaM resulted in a 75% reduction in the recovery of trimers compared to the wild type. A 6.2 kDa polypeptide was observed in the Photosystem I preparations from the wild type, but not from the psaK2 (-) strain, suggesting the presence of PsaK2 in the Photosystem I complexes. Using reverse-transcription and polymerase chain reaction, we confirmed the expression of the psaK2 gene in Synechocystis sp. PCC 6803. To conclude, both psaK1 and psaK2 are expressed in Synechocystis sp. PCC 6803 and the absence of both proteins results in only a small reduction in Photosystem I electron transport. The PsaM subunit is required for the formation of stable Photosystem I trimers.