The causes of the variation between genomes in their guanine (G) and cytosine (C) content is one of the central issues in evolutionary genomics.The thermal adaptation hypothesis conjectures that, as G:C pairs in DNA are more thermally stable than adenonine:thymine pairs, high GC content may be a selective response to high temperature. A compilation of data on genomic GC content and optimal growth temperature for numerousprokaryotes failed to demonstrate the predicted correlation. By contrast, the GC content of structural RNAs is higher at high temperatures. The issue that we address here is whether more freely evolving sites in exons (i.e. codonic third positions) evolve in the same manner as genomic DNA as a whole, showing no correlated response, or like structural RNAs showing a strong correlation. The latter pattern would provide strong support for the thermal adaptation hypothesis, as the variation in GC content between orthologous genes is typically most profoundly seen at codon third sites (GC3). Simple analysis of completely sequenced prokaryotic genomes shows that GC3, but not genomic GC, is higher on average in thermophilic species. This demonstrates, if nothing else, that the results from the two measures cannot be presumed to be the same. A proper analysis, however, requires phylogenetic control. Here, therefore, we report the results of a comparative analysis of GC composition and optimal growth temperature for over 100 prokaryotes. Comparative analysis fails to show, in either Archea or Eubacteria, any hint of connection between optimal growth temperature and GC content in the genome as a whole, in protein–coding regions or, more crucially, at GC3. Conversely, comparable analysis confirms that GC content of structural RNA is strongly correlated with optimal temperature. Against the expectations of the thermal adaptation hypothesis, within prokaryotes GC content in protein–coding genes, even at relatively freely evolving sites, cannot be considered an adaptation to the thermal environment.