The fiber-associated rumen bacterial community was phylogenetically examined by analysis of 16S rRNA gene (16S rDNA) sequences. Hay stems of orchardgrass and alfalfa were incubated for 6 and 20 h, respectively in the rumen of two different sheep, and total DNA was extracted from the incubated stems to clone bacterial 16S rDNAs using polymerase chain reaction (PCR). Of 91 such clones, 21 showed more than 97% sequence similarity with known isolates, 32 clones had 90-97% similarity with known sequences, and for the remaining 38 clones, the similarity was less than 90%. The majority of clones fell into the Cytophaga-Flavobacter-Bacteroides and low G+C Gram-positive bacterial phyla (43 and 44%, respectively). Prevotella-related and Butyrivibrio fibrisolvens-related sequences formed large clusters in the phylogenetic tree. Unknown sequences were found to form three unique clusters, one of which was suggested by semi-quantitative PCR to be more prevalent in the rumen receiving a high alfalfa diet.