The rnpB gene is universally present in bacterial species and encodes the RNA subunit of endoribonuclease P. In this study, rnpB was sequenced in 50 type strains and 29 additional strains of the genus Streptococcus. Putative secondary-structure models and possible interactions in RNase P RNA molecules are discussed. Phylogenetic relationships were studied and Bayesian, maximum-parsimony and minimum-evolution analyses supported six main clades that comprised 22 of the 50 species analysed. Phylogenetic inference was also studied for the 16S rRNA gene; it indicated a similar tree topology, but with weaker support values than for rnpB. Combined analysis of rnpB and 16S resulted in a phylogeny with significantly better support. Variability in the rnpB and 16S genes among all type strains, calculated as Shannon-Wiener information index values, was 0.45 for rnpB and 0.15 for 16S. Intraspecies proximity was assessed by principal coordinate analysis of rnpB for 32 strains of six closely related species (two clades) and showed species-specific clusters, but heterogeneity occurred in two species. It can be concluded that the rnpB gene is suitable for phylogenetic analysis of closely related taxa and has potential as a tool for species discrimination.