Cyanobacteria are often the most conspicuous structural part of microbial mats. They are also the only oxygenic phototrophs capable of N2 fixation (diazotrophy). This represents an important advantage for persistence in the often N-depleted marine intertidal microbial mats. In this study the daily pattern of expression of the structural gene for dinitrogenase reductase, nifH and the 16S rRNA gene of 3 benthic filamentous diazotrophic Cyanobacteria (Lyngbya sp., Nodularia sp. and Anabaena sp.) was measured by using quantitative RT-PCR. Gene expression patterns were compared and related to the daily pattern of nitrogenase activity. Microscopic observations revealed that the non-heterocystous Lyngbya sp. was the major cyanobacterial morphotype in all the microbial mats studied. When normalized to copy number, nifH as well as 16S rRNA gene expression by Lyngbya sp. was higher than by the heterocystous Cyanobacteria in all but one mat type. Related to the total amount of extracted RNA, Lyngbya sp. dominated 16S rRNA gene expression as well but was outcompeted by the heterocystous Cyanobacteria with respect to total nifH expression. This was due to the low DNA copy number of Lyngbya sp. nifH. The results revealed different cell-specific expression levels and varying contribution to the overall nifH and 16S rRNA gene expression by the 3 Cyanobacteria over the course of a daily cycle as well as in the different mat types. With respect to the overall nifH expression, the structurally dominant diazotroph was not the most active. Moreover, nifH expression pattern did not follow nitrogenase activity.