Summary A fragment of the small subunit rRNA gene flanked by 2 conserved Pst I sites in the 10 described Naegleria spp. and 4 clusters of the N. gruberi species complex has been sequenced. The length of the Pst I fragment varied from 794 nucleotides for N. australiensis and N. italica to 801 nucleotides for N. andersoni. The sequences of the 14 strains display a total of 154 variable positions. The number of nucleotides different between 2 strains ranges from 3 nucleotides between clusters of the N. gruberi species complex to 96 nucleotides between N. andersoni and N. jadini. In some cases the number of base substitutions between species is lower than between certain subspecies. Therefore, the subspecies are upgraded to species level. A new species name is proposed for strains previously found to be related to the former subspecies italica. Molecular phylogenies obtained with distance methods show three well separated branches: the N. fowleri — N. lovaniensis cluster, the N. andersoni — N. jamiesoni cluster and a branch with all other strains. The phylogenetic separation of the species N. jadini and N. galeacystis is confirmed by this method. On the other hand, the distances between the other species and clusters, and the bootstrap values obtained for the third branch, are too low to attribute value on their true relationship.