Factors controlling phytoplankton distribution and activity in the marine environment are often poorly understood. Knowledge of genomic content and organization may aid in understanding these processes. To this end, bulk environmental DNA was extracted from Gulf of Mexico seawater and cloned into a bacterial artificial chromosome (BAC) vector to generate a library of 3000 large-insert clones. This library was probed for various types of RubisCO gene (rbcL); 8 rbcL-containing clones (20 to 73 kb in size) were detected and sequenced. rbcL sequences indicated that they originated from marine α-Synechococcus spp. Genomic organization was highly similar to that of the sequenced genome of Synechococcus sp. strain WH 8102 in the region surrounding rbcL. Several differences from known α-Synechococcus were also observed, including putative monoamine oxidase and amino acid transport genes, and electron transport, photosynthesis and hypothetical protein genes similar to those of Prochlorococcus spp. (i.e. flavodoxin, thioredoxin reductase and iron-stress-induced chlorophyll-binding proteins). These findings indicate that the order and composition of the carbon fixation operon (cbbX to ccmK) is highly conserved in α-Synechococcus, while adjacent genomic composition indicates expanded metabolic capabilities obtained from gene-transfer events with Prochlorococcus spp. and other cyanobacteria.