Mature microbial mats from sandy intertidal beaches of the Texas Gulf coast demonstrated substantial levels of nitrogenase activity. Two species of non-heterocystous cyanobacteria, a Hydrocoleum and a Microcoleus, dominated the upper green layer of mature mats. Subsurface layers of cyanobacteria, but not mature mats, appeared during dry seasons. These "proto-mats" contained almost exclusively Microcoleus and demonstrated neither nitrogenase activity nor detectable nitrogenase reductase (Fe-protein). Hydrocoleum, identified from its morphology and 16s rDNA, was isolated and cultivated as unialgal cultures. Similar diel patterns of nitrogenase activity and Fe-protein expression were found in intact mature mats and in isolated Hydrocoleum cultures. Primers and a probe specific for the Hydrocoleum nifH gene, along with q-rtPCR measurements, demonstrated similar levels, but slightly different patterns, of expression in intact mature mats and cultures of isolated Hydrocoleum. Increased levels of nifH transcripts and Fe-protein in Hydrocoleum cultures appeared before the end of the light period of a diel cycle, and the light period was required for nitrogenase activity in the subsequent dark period. Levels of nifH transcripts stayed very low and nitrogen fixation stopped when cultures were maintained under continuous darkness. The pattern of nitrogenase activity in Hydrocoleum cultures was not affected by elimination of ambient O₂, increasing or decreasing temperature in a range from 20 ˚C to 35 ˚C, or light intensity. However, the level of nitrogenase activity did vary with environmental conditions. Highest nitrogenase activities were observed when assays were conducted in an aerobic rather than an anaerobic environment, at 25 ˚C rather than a higher or lower temperature, and illuminated with bright (~ 900 [mu]E/m²s¹), rather than less intense light. Average levels of nifH transcripts were positively correlated with levels of nitrogenase activity. Isolated cultures of Hydrocoleum formed mat-like structures in undisturbed flasks, suggesting that Hydrocoleum may be an early colonizer of intertidal sand for mat formation. However, observations of subsurface proto-mats indicate that Microcoleus is more likely to serve as the foundation for cyanobacterial mats, with Hydrocoleum later providing structural integrity and nitrogen availability. A process of successional development of microbial mats from the South Texas Gulf Coast is hypothesized. / text