The rotA gene encoding peptidyl-prolyl cis-trans isomerase has been identified, sequenced, and shown to be transcribed in the cyanobacterium Synechococcus sp. PCC7942. Inactivation of the gene by replacement of a region containing the open reading frame with a gene conferring kanamycin resistance resulted in merodiploids containing both the wild type and the modified genomic region. We were not able to isolate a kanamycin-resistant mutant in which all the genomic wild-type copies were substituted, which suggests that such replacement could have been lethal.