In many environments, biological nitrogen fixation can alleviate nitrogen limitation. The high rates of N(2) fixation often observed in cyanobacterial mats suggest that N(2) fixation may be an important source of N. In this study, organisms expressing nifH were identified in a Lyngbya sp.- and two Microcoleus chthonoplastes-dominated cyanobacterial mats. The pattern of nitrogenase activity was determined for the Lyngbya sp. mat and a Microcoleus chthonoplastes mat sampled directly in Guerrero Negro, Mexico. Their maximum rates were 23 and 15 micro mol of C(2)H(4) m(-2) h(-1), respectively. The second Microcoleus mat, which was maintained in a greenhouse facility, had a maximum rate of 40 micro mol of C(2)H(4) m(-2) h(-1). The overall diel pattern of nitrogenase activity in the three mats was similar, with the highest rates of activity occurring during the dark period. Analysis of nifH transcripts by reverse transcription-PCR revealed that several different organisms were expressing nifH during the dark period. nifH phylotypes recovered from these mats were similar to sequences from the unicellular cyanobacterial genera Halothece, Myxosarcina, and Synechocystis, the filamentous cyanobacterial genera Plectonema and Phormidium, and several bacterial nifH groups. The results of this study indicate that several different organisms, some of which were not previously known to fix nitrogen, are likely to be responsible for the observed dark-period nitrogenase activity in these cyanobacterial mats.