Almost complete (>96%) 16s rRNA sequences from nine ruminant mycoplasmas have been determined by solid-phase DNA sequencing. Polymorphisms were found in four of the 16s rRNA sequences, which indicated the existence of two different rRNA operons. Seven polymorphisms were found in Mycoplasma agalactiae, three were found in Mycoplasma bovis, one was found in Mycoplasma alkalescens, and one was found in Mycophinu bovirhinis. The sequence data were used for construction of phylogenetic trees. All but one of the ruminant mycoplasmas sequenced in this work clustered in the hominis group. A close relationship was found between M. agalactiae and M. bovis, with a 99% nucleotide similarity between their 16s rRNA sequences. They were also found to be members of the Mycoplasma lipophilum cluster of the hominis group. Furthermore, the 16s rRNA comparisons showed that Mycoplasma alkalescens and Mycoplasma canadense are closely related (>98.5%), and these species were found to cluster in the Mycoplasma hominis cluster of the hominis group. Interestingly, M. bovirhinis grouped in a new phylogenetic cluster of the hominis group. The new cluster, which was supported by bootstrap percentage values, signature nucleotide analysis, and higher-order structural elements, was named the Mycoplasma synoviae cluster. Mycoplasma bovoculi, Mycoplasma conjunctivae, and Mycoplasma ovipneumoniae clustered in the Mycoplasma neurolyticum cluster of the hominis group. Mycoplasma alvi clustered with Mycoplasma pirum in the M. pneumoniae cluster of the pneumoniae group.Mycoplasmas (class Mollicutes) are among the smallest microorganisms capable of self-replication and autonomous life. About 150 species are known today, and they are closely related to gram-positive bacteria with a low G+C content in the genome (17). Many mycoplasmas are pathogenic for animals, and they are therefore of great concern in veterinary medicine (18,20). Most mycoplasmas are host specific, and ruminants harbor a number of interesting species. Two very important and closely related species are Mycoplasma bovis and Mycoplasma agalactiae. The former causes calf pneumonia, mastitis, and arthritis in cattle (3,21), and the latter species is the causal agent of contagious agalactia in goats and sheep (10). The other species used for 16s rRNA sequencing in this study are also of clinical significance, except Mycoplasma alvi. Mycoplasma alkalescens causes arthritis in cattle. M. alvi has no known disease-causing potential, but has been isolated from the respiratory tract of cattle. Mycoplasma bovirhinis is often isolated from cattle with respiratory disease, but is probably not a primary pathogen. Mycoplasma bovoculi is associated with conjunctivitis and keratoconjunctivitis in cattle and is often found in the eyes of calves. Mycoplasma canadense is believed to cause mastitis in cattle. Mycoplasma conjunctivae is the causal agent of keratoconjunctivitis in goats and sheep, but it is not clear whether it is a primary or a secondary pathogen. Mycoplasma ovipneumoniae is by far the mos...