Random gene tagging was used to obtain new mutants of the marine cyanobacterium, Synechococcus sp. PCC7002, with defects in the CO2-concentrating mechanism (CCM). Two of these mutants, K22 and A41, showed poor growth at limiting CO2. Isolation and sequencing of a 6. 6 kb genomic region revealed the existence of five potential protein-coding regions, all arranged in the same transcriptional direction. These regions code for an RbcR homologue, NdhF3 (subunit 5 of type 1 NAD(P)H dehydrogenase; NDH-1 complex), NdhD3 (subunit 4 of NDH-1), ORF427 and ORF133 (hypothetical proteins). Insertional mutants in ndhD3, ndhF3 and ORF427, like A41 and K22, were all incapable of inducing high-affinity CO2 uptake and were not fully capable of inducing high-affinity HCO3- transport. ndhD3 and ndhF3 mutants displayed P700 re-reduction rates identical to wild-type cells, suggesting that NdhD3 is part of a specific NDH-1 complex that is not involved in photosynthetic cyclic electron transport. Thus, it is feasible that NdhD3, NdhF3 and ORF427 might form part of a novel NDH-1 complex located on the cytoplasmic membrane and involved in tightly coupled energization of high-affinity CO2 transport. The possibility of multiple, functionally distinct NDH-1 complexes in cyanobacteria is discussed.