A thermophilic microbial mat with a relatively simple morphological composition was used to study the expression of key metabolic genes between mat layers. Mats comprised Roseiflexus castenholzii, Synechococcus sp., a Sphingomonas-like proteobacterial taxon and an unidentified member of the Thermotogae as determined by 16S rRNA phylotypes. The diversity of expressed loci for key genes involved in oxygenic photosynthesis (cbbL), anoxygenic photosynthesis (pufM) and nitrogen fixation (nifH) was assessed. The cyanobacterial surface layer supported two cbbL transcripts, with closest phylogenetic affinity to those from the cyanobacterium Synechococcus sp. and a proteobacterium Nitrobacter sp. This indicates that both photoautotrophic and chemolithoautotrophic carbon dioxide fixation may occur in this mat layer. Lower layers did not support cbbL transcripts. Anoxygenic photosynthesis was indicated by a single pufM transcript with closest affinity to that of R. castenholzii. Expression occurred in all layers beneath the cyanobacterial surface layer. Expression of a single nifH transcript with closest affinity to a proteobacterial nitrogenase occurred in samples throughout all mat layers.