Molecular diversity of rumen bacteria was analyzed by PCR amplification and sequencing of 16S rDNA clone libraries prepared from the rumen content of Holstein cows fasted for 16 h. A total of 84 clones, containing almost full size 16S rDNA sequences (about 1.5 kb long), were completely sequenced and subjected to an on line similarity search. Four sequences from the 84 clones closely resembled that of Butyrivibrio fibrisolvens and one clone was found to be related to Treponema bryantii. For 38% of the sequences, the similarity with database sequences was in the range of 90%–98% and for the remaining 56% the similarity was less than 90%. The bacterial community structure was also revealed by phylogenetic placement of sequences in relation to different fractions of rumen content. In the library from the rumen fluid, the sequences were affiliated with the following major phyla: low G+C Gram-positive bacteria (52.4%), Cytophaga-Flexibacter-Bacteroides (38.1%), Proteobacteria (4.7%) and Spirochaetes (2.4%). 2.4% had an uncertain affiliation. The vast majority of sequences from the rumen solids were found to be related to low G+C Gram-positive bacteria (71.4%) and the remaining sequences were placed within the Cytophaga-Flexibacter-Bacteroides (26.2%) and Spirochaetes (2.4%) phyla.