A naturally occurring atrazine-resistant cyanobacterial isolate, strain SG2, was isolated from an atrazinecontaining wastewater treatment system at the Syngenta atrazine production facility in St. Gabriel, La. Strain SG2 was resistant to 1,000 μg of atrazine per ml but showed relatively low resistance to diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl urea]. Analyses of 16S ribosomal DNA indicated that strain SG2 falls into the Synechocystis/Pleurocapsa/Microcystis group. Photosynthetically driven oxygen evolution in strain SG2 was only slightly inhibited (about 10%) by 2,000 μg of atrazine per ml, whereas in the control strain Synechocystis 6803, oxygen evolution was inhibited 90% by 1,000 μg of atrazine per ml. No atrazine accretion, mineralization, or metabolites were detected when strain SG2 was grown with [14C]atrazine. Strain SG2 contained three copies of the psbA gene, which encodes the D1 protein of the photosystem II reaction center. Nucleotide sequence analyses indicated that the psbA2 and psbA3 genes encoded predicted proteins with the same amino acid sequence. However, the psbA1 gene product contained five extra amino acids, which were not found in PsbA proteins from five other cyanobacteria. Moreover, the PsbA1 protein from strain SG2 had an additional 13 amino acid changes compared to the PsbA2/PsbA3 proteins and contained 10 amino acid alterations compared to conserved residues found in other cyanobacteria. Reverse transcriptase PCR analysis indicated that the psbA1 gene and the psbA2/psbA3 gene(s) were expressed in photosynthetically grown cells in the presence of atrazine. These results suggest that strong selection pressure conferred by the continual input of atrazine has contributed to the evolution of a herbicide-resistant, yet photosynthetically efficient, psbA gene in a cyanobacterium.