Some of the wall-less bacteria of the class Mollicutes (mycoplasmas) have pyrophosphate (PP,)-dependent enzymic activities, including PP,-dependent phosphofructokinase (PP,-PFK) , PP,-dependent nucleoside kinase, and pyruvate,orthophosphate dikinase (PPDK) activities. In most other bacteria, adenosine 5'-triphosphate (ATP), not PP,, is the cofactor of analogous enzymic reactions. Because PP,-dependent enzymes are more common among mollicutes than other bacteria, we describe here an examination of the six walled bacteria that have been reported to be phylogenetically related to the mollicutes (Clostridium innocuum, Clostridium ramosum, Erysipelothrix rhusiopathiae, Lactobacillus catenaformis, Lactobacillus vitulinus, and Streptococcus pleomorphus) for PP,-PFK, ATP-dependent PFK, phosphoenolpyruvate carboxytransphosphorylase, PPDK, and PP,-and ATP-dependent acetate kinases. Two anaerobic mollicutes, Anaeroplasma intermedium and Asteroleplasma anuerobium, were also tested. C. innocuum, E. rhusiopathiue, S . pleomorphus, and Anueroplasma intermedium had PPi-PFK activities, whereas C . ramosum, the two lactobacilli, and Asteroleplasma anaerobium had only ATP-dependent PFK activities. Asteroleplasma anaerobium and all of the walled bacteria except E. rhusiopathiue had PPDK activities. All of the species except Asteroleplasma anaerobium and E . rhusiopathiae also had pyruvate kinase activities; the effects of allosteric activators were tested. Phosphoenolpyruvate carboxytransphosphorylase was detected by using two methods in C . innocuum, C . ramosum, and S . pleomorphus. All of the species tested had ATP-dependent acetate kinase activities, but none had detectable PP,-dependent acetate kinase activity. The occurrence of one or more PPi-dependent enzymes in the mollicutes and their walled relatives is a phenotypic indicator of the phylogenetic relatedness of these organisms. The distribution of these enzymes among members of this group substantiates the subgroups proposed by other workers who used 16s ribosomal ribonucleic acid analysis.The wall-less eubacteria belonging to the genera Mycoplasma, Ureaplasma , Spiroplasma, Acholeplasma, Anaeroplasma, and Asteroleplasma are grouped together in the class Mollicutes. Phylogenetic analyses of 5s and 16s ribosomal ribonucleic acid (rRNA) sequences have revealed that the mollicutes cluster with the low-guanine-plus-cytosine content subdivision of gram-positive eubacteria that includes the bacillus-lactobacillus-streptococcus branch (31, 40, 41). Among the members of the low-guanine-plus-cytosine content gram-positive eubacteria, the mollicutes are most closely related to Clostridium innocuum, Clostridium ramosum, Erysipelothrix rhusiopathiae, Lactobacillus catenaformis (Lactobacillus catenaforme), Lactobacillus vitulinus, and Streptococcus pleomorphus (16,31,[40][41][42] The mollicutes and their walled phylogenetic relatives have few phenotypic similarities. The only comparative study of the phenotypes of the mollicutes and their walled phylogenetic relatives was conducted by...