Primase, encoded by dnaG in bacteria, is a specialized DNA-dependent RNA polymerase that synthesizes RNA primers de novo for elongation by DNA polymerases. Genome sequence analysis has revealed two distantly related dnaG genes, TtdnaG and TtdnaG2, in the thermophilic bacterium T. tengcongensis. Both TtDnaG (600 amino acids) and TtDnaG2 (358 amino acids) exhibited the primase activities in vitro at a wide range of temperature. Interestingly, the template recognition specificities of these two primases were quite distinctive. When the trinucleotide-specific templates were tested, TtDnaG initiated RNA primer synthesis efficiently only on template containing trinucleotide 5'-CCC-3', but not the other 63 possible trinucleotides. When the 5'-CCC-3' was flanked with additional cytosines or guanines, the initiation efficiency of TtDnaG increased remarkably. Significantly, TtDnaG could specifically and efficiently initiate RNA synthesis on a limit set of other tetranucleotides composed entirely of cytosines and guanines, indicating that TtDnaG initiated RNA synthesis more preferably on GC-containing tetranucleotides. On the contrary, it seemed that TtDnaG2 had no specific initiation nucleotides, as it could efficiently initiate RNA primer synthesis on all templates detected so far. The DNA binding affinity of TtDnaG2 was usually 10-fold higher than that of TtDnaG, which might correlate with its high activity but low template specificity. These distinct priming activities and specificities of TtDnaG and TtDnaG2 might shed new light on the diversity of structure and function of the primases.