The complete 5S and 16S ribosomal DNA (rDNA) sequences of the facultatively chemolithotrophic bacterium Thiobacillus cuprinus and results of a comparison of these sequences with homologous sequences from several proteobacterial species supported affiliation of T. cuprinus with the beta 1 subgroup of the Proteobacteria. T. cuprinus, Thiobacillus intermedius, Thiobacillus perometabolis, and Thiobacillus thermosulfatus form a phylogenetic cluster that comprises some of the thiobacilli capable of mixotrophic growth. This cluster is related to some pseudomonads and Alcaligenes species belonging to the beta subclass. In addition, a low-frequency restriction fragment analysis (LFRFA) of some mixotrophic thiobacilli and some related species was carried out by using pulsed-field gel electrophoresis (PFGE) to determine the SpeI and XbaI macrorestriction patterns and genome sizes of these organisms. The correlation of the LFRFA results and the 16S rDNA analysis results and the usefulness of the two analyses are discussed. The PFGE fingerprints suggested that Thiobacillus sp. strain ATCC 27793 is related to T. intermedius rather than to T. perometabolis, as described previously. The distinctive characteristics of the mixotrophic species analyzed in this work, their phylogenetic relatedness, and their physiological differences from other groups belonging to the Proteobacteria, including other thiobacilli, suggest that these organisms should be transferred to a new genus, the genus Thiomonas gen. nov.