The placement of the extreme thermophile Aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. We investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically DnaG primase. Although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for A. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. To determine the full range of A. aeolicus primase activity, two oligonucleotides were designed that comprised all potential trinucleotide initiation sequences. One of the screening templates supported primer synthesis and the lengths of the resulting primers were used to predict possible initiation trinucleotides. Use of trinucleotide-specific templates demonstrated that the preferred initiation trinucleotide sequence for A. aeolicus primase was 5'-d(CCC)-3'. Two other sequences, 5'-d(GCC)-3' and d(CGC)-3', were also capable of supporting initiation, but to a much lesser degree. None of these trinucleotides were known to be recognition sequences used by other microbial primases. These results suggest that the initiation specificity of A. aeolicus primase may represent an adaptation to a thermophilic environment.