The psbL gene is a member of the psbEFLJ gene cluster in the cyanobacterium Synechocystis sp. PCC 6803 and higher plants. psbL, a 4.5 kDa protein encoded by this gene, is a component of the photosystem II complex. The amino acid sequence of this protein indicates that it has a single membrane-spanning alpha-helical domain. We have used a targeted mutagenesis technique to delete the coding region of the psbL gene in Synechocystis 6803. The resultant mutant strain T345 did not show any PSII-mediated oxygen evolution activity and, as a result, could not grow under photoautotrophic conditions. However, it had normal PSI activity. The chlorophyll to phycobilin ratio in the T345 cells was significantly lower than that in the wild type cells. Fluorescence emission spectra (77 K) of the mutant cells showed the absence of a 695 nm band that usually originates from the PSII complex. Binding assays with radioactive diuron demonstrated that the mutant cells did not have any herbicide binding activity. However, immunostaining experiments showed that both the D 1 (the herbicide binding protein) and the D2 proteins of the PSII reaction center were present at > 25% of their normal levels in the thylakoid membranes of the T345 mutant cells. Our data indicate that the PsbL protein is essential for the normal functioning of PSII.