The relationships among ant subfamilies were studied by phylogenetic analysis of rDNA sequences of 15 species from seven subfamilies. PCR primers were designed on the basis of the rDNA sequence of the Australian bulldog ant, Myrmecia croslandi, previously determined. Phylogenetic trees were constructed using sequences of a fragment of 18S rDNA (1.8 kb), a fragment of 28S rDNA (0.7 kb excluding variable regions) and a combination of the 18S and 28S rDNAs, by neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML). rDNA sequences corresponding to the same fragments from three non-ant hymenopteran species (a sawfly, a bee and a wasp) were employed as outgroups. These trees indicated that the ant subfamilies were clustered singly, and, among the seven subfamilies examined, Ponerinae and six other subfamilies are in a sister-groups relationship. The relationship among the six subfamilies, however, was not clarified. The phylogenetic trees constructed in the present study are not in contradiction to the tree from cladistic analysis of morphological data by Baroni Urbani et al. (1992) and the tree from morphological and molecular data (Ward and Brady, 2003), but are inconsistent with the traditional phylogeny. The present results thus raise a question as to the status of some traditionally employed "key" morphological characters. The present results also call for a reexamination of Amblyopone traditionally treated as a member of Ponerinae as belonging to a new subfamily.